Title:
CRYSTALS OF MEMBRANE PROTEINS
Kind Code:
A1


Abstract:
A polypeptide in crystalline form comprises a G-protein coupled receptor (GPCR) with an IC3 loop substituted by an amino acid residue sequence of lysozyme.



Inventors:
Stevens, Raymond C. (La Jolla, CA, US)
Hanson, Michael A. (San Marcos, CA, US)
Cherezov, Vadim (San Diego, CA, US)
Kuhn, Peter (Solana Beach, CA, US)
Application Number:
13/852401
Publication Date:
08/15/2013
Filing Date:
03/28/2013
Assignee:
The Scripps Research Institute (La Jolla, CA, US)
Primary Class:
Other Classes:
435/206
International Classes:
C07K14/72; C12N9/24; C12N9/96
View Patent Images:



Other References:
Wiencek, J. M. Ann. Rev. Biomed. Eng. 1999, 1, 505-534.
Martin Caffrey Membrane protein crystallization. J. Struc. Biol. (2003) 142, 108-132.
Primary Examiner:
NASHED, NASHAAT T
Attorney, Agent or Firm:
Olson & Cepuritis, LTD. (CHICAGO, IL, US)
Claims:
We claim:

1. A composition comprising a polypeptide in crystalline form, wherein said polypeptide comprises: a G-protein coupled receptor (GPCR) comprising an IC3 loop containing a substitution that comprises the amino acid sequence of a lysozyme.

2. A composition comprising a fusion protein in crystalline form, wherein said fusion protein comprises, from N-terminus to C-terminus: a) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises TM1, TM2, TM3, TM4 and TM5 regions of said GPCR; b) a domain comprising the amino acid sequence of a lysozyme; c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR.

3. The composition of claim 2, wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occurring GPCR.

4. The composition of claim 1, wherein said GPCR is a β2-adrenergic receptor.

5. The composition of claim 2, wherein said fusion protein is bound to a ligand for said GPCR.

6. A composition comprising a polypeptide in crystalline form, wherein said polypeptide comprises, from N-terminus to C-terminus: a) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises the amino acid sequence that is N-terminal to the IC3 loop of said GPCR; b) a domain comprising the amino acid sequence of a lysozyme; c) a second portion of said GPCR, wherein said second portion comprises the amino acid sequence that is C-terminal to the IC3 loop of said GPCR.

7. A crystalline fusion protein which comprises a beta-2 adrenergic receptor and lysozyme.

8. The crystalline fusion protein of claim 7 wherein the lysozyme is a T4 lysozyme sequence.

9. The crystalline fusion protein of claim 7 wherein the beta-2 adrenergic receptor has a E122W mutation.

10. The crystalline fusion protein of claim 7 wherein the beta-2 adrenergic receptor has a E122W mutation and wherein the lysozyme is a T4 lysozyme sequence.

11. The crystalline fusion protein of claim 7 wherein the lysozyme is T4 lysozyme sequence in place of the third intracellular loop of the beta-2 adrenergic receptor.

12. The crystalline fusion protein of claim 7 wherein residues 2315.70 to 2626.24 are replaced by residues 2 to 161 of T4 lysozyme, and the beta-2 adrenergic receptor is truncated at position 365.

13. The crystalline fusion protein of claim 7 further comprising a ligand non-covalently bound thereto.

14. The crystalline fusion protein of claim 13 wherein the ligand is carazolol.

Description:

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/199,611 filed on Sep. 2, 2011 which is a continuation of U.S. Ser. No. 12/739,134 filed on Sep. 23, 2010 which is a U.S. National Stage of PCT/US2008/080847, filed Oct. 22, 2008, and claims benefit of U.S. provisional application 60/999,951, filed Oct. 22, 2007; U.S. provisional application 61/000,325, filed Oct. 24, 2007; U.S. provisional application 61/060,107, filed Jun. 9, 2008; and U.S. provisional application 61/194,961, filed Oct. 1, 2008, each of which is incorporated herein by reference, in its entirety, for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. GM73197 awarded by the National Institutes of Health; GM74691 and GM62411 awarded by the Protein Structure Initiative; Y1-CO-1020 awarded by the National Cancer Institute; and Y1-GM-1104 awarded by the National Institute of General Medical Sciences. The government has certain rights in this invention.

Coordinates and structure factors have been deposited in the Protein Data Bank with identification code 2RH1.

BACKGROUND OF THE INVENTION

Naturally occurring polypeptides or proteins often fold into complex, three-dimensional shapes that determine both chemical and physiological functionality. Thus a thorough understanding of proteins necessarily involves a detailed representation of their spatial topography. The field of protein crystallography has flourished over the last 20 years resulting in a rapid increase in the knowledge bas of protein structure enabling great strides in other disciplines including biochemistry, pharmaceutical development and cell biology. However, the structural biology field has largely been restrained to working with protein that is naturally soluble in aqueous media, or made soluble by incorporation into surfactant micelles. The present invention provides methods and compositions that allow for the study of membrane-embedded proteins (i.e., integral membrane proteins) in a more natural membrane bilayer environment. The present invention enable a more detailed analysis of important classes of membrane-embedded polypeptides that play key roles in a variety of cellular processes including energy and signal transduction.

SUMMARY OF THE INVENTION

Other objects, features and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference for all purposes and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

In one aspect the invention provides compositions, e.g., useful for the crystallization of membrane protein. In some embodiments, the composition is suitable for lipidic cubic phase crystallization. In some embodiments the compositions contain 10-60% v/v of a polyethylene glycol, 0.01-0.5 M of a salt, 1-20% v/v of an organic compound, and 1-50% w/w of a lipid additive in a host lipid. In some embodiments of the compositions, the protein to be crystallized is present at a concentration of 1 to 100 mg/mL. In some embodiments, the protein to be crystallized is present at a concentration of 50 mg/mL.

In some embodiments of the compositions of the inventions, the polyethylene glycol is PEG or modified PEG at a molecular size of 10-8000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-8000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-2000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-1000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400. Examples of modified PEG include but are not limited to PEG laurate, PEG dilaurate, PEG oleate, PEG dioleate, PEG stearate, PEG distearate, PEG glyceryl trioleate, PEG glyceryl laurate, PEG glyceryl stearate, PEG glyceryl oleate, PEG palm kernel oil, PEG hydrogenated castor oil, PEG castor oil, PEG corn oil, PEG caprate/caprylate glycerides, PEG caprate/caprylate glycerides, PEG cholesterol, PEG phyto sterol, PEG soya sterol, PEG trioleate, PEG sorbitan oleate, PEG sorbitan laurate, PEG succinate, PEG nonyl phenol series, PEG octyl phenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHS Ester and methoxypoly(ethylene glycol) (mPEG).

In some embodiments of the compositions, the salt is a sulfate salt. In some embodiments, the sulfate salt is sodium sulfate. In some embodiments, the salt is present at a concentration of 0.1-0.5 M. In some embodiments, the salt is present at a concentration of 0.1-0.2 M. In some embodiments, the compositions of the invention contain a buffer. In some embodiments, the buffer is present at a concentration of 0.01-0.5 M. In some embodiments, the buffer is present at a concentration of 0.1-0.2 M. In some embodiments, the buffer is present at a concentration of 0.1 M. In some embodiments, the buffer is Bis-tris propane. In some embodiments, the buffer has a pH 6.5-7.0. In some embodiments of the compositions, an organic compound is present at a concentration of 1-10% v/v. In some embodiments, the organic compound is present at a concentration of 5-7% v/v. In some embodiments, the organic compound is 1,4-butanediol.

In some embodiments of the compositions, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of lipid additives include but are not limited to cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments, the host lipid is monoolein.

In some embodiments of this aspect, the composition of the invention comprises 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid.

In another aspect the invention includes compositions suitable for lipidic cubic phase crystallization. In some embodiments, the compositions of the inventions contain a lipid additive. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.

In another aspect the invention includes methods for crystallization of membrane proteins. In some embodiments, the method for crystallization of membrane proteins comprises adding a lipid additive to a lipidic cubic phase. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.

In another aspect, the invention provides for methods of crystallization of a protein. In some embodiments of this aspect, the method comprises, providing said protein in a lipidic cubic phase composition, filling a plate comprising a material that does not interfere with imaging (such as, e.g., a transparent glass or plastic) with said composition, placing said plate containing said composition under conditions suitable for crystallization of said protein and detecting the presence of a crystal of said protein in said plate. In some embodiments, the method further comprises covering said plate with a second plate comprising a material that does not interfere with imaging (such as, e.g., a transparent glass or plastic).

In some embodiments of the methods, the protein is a non-colored protein. In some embodiments, the protein is a G protein-coupled receptor (GPCR). In some embodiments, the protein comprises a β2AR, a CXCR4, or a human adenosine A2A receptor. In some embodiments the protein comprises a stabilizing point mutation or a T4 lysozyme fusion or both.

In some embodiments of the methods, the lipidic cubic phase composition comprises a lipid additive. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.

In some embodiments of the methods, the first plate and second plate are made of glass. In some embodiments, the plate allows for the control of crystallization conditions, such as the humidity and temperature of said lipidic cubic phase composition.

In some embodiments of the methods, the crystals are harvested directly from the plate. In some embodiments of the methods, the crystals are harvested between the cubic and the sponge phase of the lipidic cubic phase composition. In some embodiments of the methods, the crystals are harvested directly from said lipidic cubic phase composition and placing said crystals in liquid nitrogen.

In another aspect the invention provides methods for screening a crystal present in a liquid cubic phase composition. In some embodiments, the method comprises exposing the composition to a first beam and determining a change of the first beam, exposing the composition to a second beam and determining a change of the second beam, and identifying an area where the crystal is present in said composition. Examples of changes in the beams, include but are not limited to, change in direction and/or intensity of the beams. In some embodiments the crystals are non-colored.

In some embodiments of the methods, the first beam and second beam are attenuated. In some embodiments the beams are attenuated 10 times. In some embodiments, the first beam is a stilted 100×25 μm beam. In some embodiments, the methods comprise exposing said lipidic cubic phase composition to a third beam. In some embodiments, the methods comprise exposing said lipidic cubic phase composition to up to ten extra beams. In some embodiments, the exposure of composition to the beams is 2 seconds. In some embodiments, the beams are beams of visible light.

In another aspect the invention includes a crystal of a membrane protein. In some embodiments, the invention includes a crystal of a G protein-coupled receptor (i.e., a “GPCR”) non-covalently bound to a ligand. In some embodiments, the extracellular domain of said crystalline GPCR is resolvable by X-Ray crystallography. In some embodiments, the ligand is a diffusible ligand.

In some embodiments of this aspect, the volume of the crystal is greater than 15×5×1 μm. In some embodiments, the volume of said crystal is greater than 30×5×5 μm. In some embodiments of this aspect, the volume of crystal is greater than 40×20×5 μm. In some embodiments, volume of the crystal is estimated assuming that each of the stated dimensions are orthogonal so that the volume estimate is the product of the dimensions. In some embodiments, the crystal is suitable for X-ray crystallography. In some embodiments, X-ray crystallographic analysis can be carried out to determine the structure of a protein comprising said crystal.

In some embodiments of this aspect, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments, the crystal diffract to a resolution of 1.0 to 10.0 Å. In some embodiments, the crystal diffract to a resolution of 2.0 to 5.0 Å. In some embodiments, the crystal diffract to a resolution of 2.2 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 3.2, 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.4 Å.

In some embodiments, the G protein-coupled receptor is a β2AR protein, a CXCR4 protein, or a human adenosine A2A receptor protein.

In another aspect the invention provides for a crystal of β2AR. In some embodiments, the structure of an extracellular domain of said crystal is capable of being resolved by X-ray crystallography. In some embodiments, the crystal comprises 442 amino acids, a palmitic acid covalently bound to Cys341 and an acetamide molecule bound to Cys2656.27, a diffusible ligand, up to 10 molecules a lipid additive, up to five salt ions and up to 10 butanediol molecules. In some embodiments, the lipid additive is cholesterol. In some embodiments, the crystal comprises three cholesterol molecules. In some embodiments, the salt ion is a sulfate ion. In some embodiments, the crystal comprises two sulfate ions. In some embodiments, the diffusible ligand is carazol. In some embodiments the crystal comprises two butanediol molecules. In some embodiments of this aspect, the volume of the crystal is greater than 15×5×1 μm. In some embodiments, the volume of said crystal is greater than 30×5×5 μm. In some embodiments of this aspect, the volume of the crystal is greater than 40×20×5 μm. In some embodiments the volume of the crystal is estimated by assuming that each dimension is orthogonal to the other dimensions so that the volume is the product of the three lengths. In some embodiments, the crystal is suitable for X-ray crystallography. In some embodiments, the structure of a β2AR protein can be determined from said crystal using X-ray crystallographic analysis. In some embodiments, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments of this aspect, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments, the crystal diffract to a resolution of 1.0 to 10.0 Å. In some embodiments, the crystals diffract to a resolution of 2.0 to 5.0 Å. In some embodiments, the crystal diffract to a resolution of 2.2 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 3.2, 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.4 Å.

In another embodiment, the invention provides a composition for lipidic cubic phase crystallization of a membrane protein, comprising a polyethylene glycol or modified polyethylene glycol; 0.01-1M of a salt; a host lipid; a lipid additive, wherein said lipid additive is present at 10-60% v/v ratio relative to the host lipid; a buffer; and 1 to 100 mg/ml of a membrane protein. In a related embodiment, the polyethylene glycol is PEG or modified PEG, wherein said PEG or modified PEG has an average molecular weight of 200-20,000, 400-8000, or 400-2000. In yet another related embodiment, the PEG or modified PEG in the composition has an average molecular weight of 400. In another related embodiment, the salt is selected from the group consisting of a sulfate salt, a citrate salt, a malonate salt, a tartrate salt, an acetate salt, and a formate salt. In certain embodiments of the composition, the salt is present at a concentration of 0.1-0.2 M. In another related embodiment, the buffer is present at a concentration of 0.05-0.5 M in the composition. In certain embodiments, the buffer is Bis-tris propane or sodium citrate. In other related embodiments of the composition, the buffer has a pH between 4.5-8.0.

In still other related embodiments of the composition for lipidic cubic phase crystallization of a membrane protein, the composition further comprising an alcohol present at a concentration of 1-10% v/v or 5-7% v/v. In certain embodiments, the alcohol is a diol or triol. In other embodiments, the alcohol is 1,4-butanediol or 2,6-hexanediol.

In still other related embodiments of the composition for lipidic cubic phase crystallization of a membrane protein, the lipid additive is present at a concentration of 1-20% w/w in a host lipid or 8-10% w/w in a host lipid. In yet another related embodiment of the composition, the lipid additive is selected from the group consisting of 2-monoolein, phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid, dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoyl phosphatidylcholine (“DOPE”), Asolectin, and a sterol. In still other embodiments, the lipid additive is a sterol. In related embodiments, the lipid additive is cholesterol.

In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein, the host lipid is selected from the group consisting of monopalmitolein, monovaccenin and monoolein. In a related embodiment, the host lipid is monoolein. In still another related embodiment, the membrane protein to be crystallized in said composition is present at a concentration of 1 to 100 mg/mL. In yet another embodiment, the membrane protein to be crystallized in said composition is present at a concentration of 40-60 mg/mL.

In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein membrane protein is a G-protein coupled receptor, such as a β2AR protein, a human adenosine A2A receptor protein, a CXCR4-T4L protein, or a (β2AR-T4L protein. In related embodiments, the G-protein coupled receptor is a protein comprising or consisting of a β2AR(E122W), a β2AR(E122W)-T4L, a human adenosine A2A receptor-T4L, a CXCR4-T4L or β2AR-T4L. In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein, the composition comprises a ligand selected from the group consisting of carazolol, timolol, alprenolol, and clenbutorol.

In another embodiment, the invention provides a method of generating crystals of a membrane protein comprising: mixing a lipid additive with a host lipid to form a lipid mixture, wherein said lipid additive is selected from the group consisting of a sterol, DOPE, DOPE-Me, DOPC, and Asolectin, and wherein said lipid additive is 5 to 20% w/w in said host lipid; and combining said lipid mixture with a membrane protein solution under conditions suitable for the formation of a lipidic cubic phase composition. In a related embodiment of the method, said protein is a non-colored protein. In a related embodiment, the amount of said lipid additive is 8 to 10% w/w in said lipid. In another related embodiment, the invention further comprises: filling a plate with said lipidic cubic phase composition, wherein said plate is compatible with imaging; placing said plate containing said lipic cubic phase composition under conditions suitable for crystallization of said protein; and detecting the presence of a crystal of said protein in said plate. In another embodiment, the method further comprises covering said plate with a second plate.

In a related embodiment of the method of generating crystals of a membrane protein, the protein is a GPCR. In yet another related embodiments, the protein comprises a β2AR. In yet another related) embodiment, the. β2AR protein is selected from the group consisting of β2AR(E122W), β2AR(E122W)-T4L, and β2AR-T4L. In yet another related embodiment, the GPCR is a human adenosine A2A receptor or a CXCR4 receptor where the proteins may comprise, in still other related embodiments, a T4 lysozome.

In yet another related embodiment of method of generating crystals of a membrane protein, the lipid additive is present at a concentration of 1-20% w/w or 8-10% w/w in a host lipid. In yet another related embodiment of the method, the second plate comprises a glass. In yet another related embodiment, the method further comprises harvesting crystals directly from said plate. Another related embodiment of the method comprises harvesting crystals from between the cubic and the sponge phase of said lipidic cubic phase composition. In another related embodiment, the method comprises harvesting crystals directly from said lipidic cubic phase composition and placing said crystals in liquid nitrogen. In yet another related embodiment, the method comprising a step of soaking into said crystal a diffusible ligand or candidate ligand.

The invention also provides a method of screening a crystal of a GPCR present in a liquid cubic phase composition comprising: preparing a liquid cubic phase composition comprising a GPCR protein, a host lipid, and a lipid additive; exposing said composition to a first X-ray beam and determining a change in direction or intensity of said first X-ray beam; exposing said composition to a second beam and determining a change in direction or intensity of said second X-ray beam; identifying an area where said GPCR crystal is present in said composition; and exposing said identified area to at least a third X-ray beam. In a related embodiment, the first beam is a slitted 100×25 μm beam. In another related embodiment, the crystal is colorless. In yet another embodiment, the GPCR crystal is β2AR(E122W)-T4L, β2AR(E122W), β2AR, or β2AR-T4L protein. In related embodiments, the crystal is human adenosine A2A receptor or a CXCR4 receptor where the proteins may comprise, in still other related embodiments, a T4 lysozome.

In another embodiment, the invention provides a crystal of a human β2AR protein wherein the extracellular loop ECL2 of said β2AR is sufficiently ordered to produce interpretable electron density in a crystallographically-derived electron density map. In yet another related embodiment, each β2AR molecule in said crystal comprises three non-covalently bound cholesterol molecules and at least one salt ion. In yet another related embodiment, the at least one salt ion is a sulfate ion. In yet another related embodiment, each β2AR molecule in said crystal said crystal comprises two sulfate ions. In yet another related embodiment, each β2AR molecule in said crystal said crystal further comprises carazol. In yet another related embodiment of the crystal, each β2AR molecule in said crystal comprises between 1 and 10 butanediol molecules. In yet another related embodiment of the crystal, the volume of said crystal exceeds 15×5×1 μm, 30×5×5 μm or 40×20×5 μm. In yet another related embodiment, the specific surface area of the crystal is 0.0001-5 m2/g. In yet another related embodiment, the crystal is crystallized using liquid cubic phase crystallization. In yet another related embodiment, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In yet another related embodiment, the crystal diffracts to a resolution of 2.0 to 10.0 Å, 2.0 to 5.0 Å, or 2.2 to 2.4 Å. In yet another related embodiment, the structure of said crystal is solved and refined at a resolution higher than about 3.2, higher than about 2.8, or higher than about 2.4 Å.

In yet another embodiment, the invention provides a crystalline form of a human β2AR protein having an atomic arrangement of coordinates comprising the β2AR coordinates set forth in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance).

In another embodiment, the invention provides a crystalline form of a human β2AR protein, where said form has unit cell dimensions of a=106.3 Angstroms, b=169.2 Angstroms, and c=40.2 Angstroms. In arelated embodiment, said β2AR protein is β2AR-T4L. In another related embodiment, the β2AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of a human β2AR protein, wherein said space group of said crystalline form is C2. In a related embodiment, said β2AR protein is β2AR-T4L. In another related embodiment, the β2AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of a human β2AR protein, wherein said crystalline form diffracts X-rays to a resolution of 2.4 Angstroms. In a related embodiment, said β2AR protein comprises a point mutation that stabilizes said β2AR protein. In another related embodiment, said β2AR protein is β2AR-T4L. In a related embodiment, the β2AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of a human β2AR protein wherein each β2AR molecule in said crystal comprises 442 amino acids, a palmitic acid covalently bound to Cys341, an acetamide molecule bound to Cys2656.27, a diffusible ligand, one to ten molecules of a lipid additive, one to five salt ions and one to ten butanediol molecules.

In another embodiment, the invention provides a method of identifying a compound that binds to a ligand binding site of a human β2AR protein, comprising comparing a set of three-dimensional structures representing a set of candidate compounds with a three-dimensional molecular model of said ligand binding site, comprising: receiving a three-dimensional model of a ligand binding site on said human β2AR protein, wherein said three-dimensional model of said ligand binding site comprises atomic co-ordinates for a plurality of ligand-binding residues, wherein said atomic co-ordinates are taken from Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance); determining, for each of the set of compound three-dimensional models, a plurality of distance values indicating distances between said atomic co-ordinates of said candidate compound of the set of candidate compounds and said atomic coordinates of said ligand-binding residues comprising said ligand binding site; determining, for each of the set of compound three-dimensional models, a binding strength value based on the plurality of distance values determined for the compound three-dimensional model, wherein the binding strength value indicates the stability of a complex formed by said human β2AR protein and a compound represented by the compound three-dimensional model; and storing a set of results indicating whether each candidate compound binds to the three-dimensional model based on the binding strength values. In a related embodiment, said ligand-binding residues comprise a plurality of residues selected from the group consisting of Y199, A200, S204, T118, V117, W286, Y316, F290, F289, N293, W109, F193, and Y308. In another related embodiment, said ligand-binding residues comprise a plurality of residues selected from the group consisting of W109, V117, T118, F193, Y199, A200, W286, F289, F290, Y316. In another related embodiment of the method of identifying a compound that binds to a ligand binding site of a human β2AR protein, said binding strength value is based on one or more of a hydrogen bonding strength, a hydrophobic interaction strength, or a Coulombic interaction binding strength. In another related embodiment, one or more of said receiving, determining, or storing steps is carried out using a commercially-available software program. In yet another related embodiment, the commercially-available software program is selected from the group consisting of DOCK, QUANTA, Sybyl, CHARMM, AMBER, GRID, MCSS, AUTODOCK, CERIUS II, Flexx, CAVEAT, MACCS-3D, HOOK, LUDI, LEGEND, LeapFrog, Gaussian 92, QUANTA/CHARMM, Insight II/Discover, and ICM. In yet another related embodiment, the method further comprising the step of contacting a human β2AR protein with a molecule comprising an identified candidate compound. In yet another related embodiment, the molecule further comprises a moiety capable of competitively displacing a ligand from said human β2AR protein, wherein said ligand binds to said ligand binding site of said human β2AR protein. In yet another related embodiment, the method further comprising characterizing a binding interaction between said human β2AR protein and said molecule comprising said identified candidate compound, and storing a result of said characterizing. In yet another related embodiment, said characterization comprises determining an activation of a function of said human β2AR protein, an inhibition of a function of said human β2AR protein, an increase in expression of said human β2AR protein, a decrease in expression of said human β2AR protein, a displacement of a ligand bound to said ligand binding site, or a stability measure for said human β2AR protein.

In another embodiment, the invention provides a method for selecting a library of potential modulators of β2AR to be screened, comprising calculating a structure of a first potential modulator using at least a portion of the structure co-ordinates of Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance), correlating said structure of said first potential modulator with a library of modulators identified as comprising said structure said first potential modulator, and storing or transmitting information about the identified library.

In yet another embodiment, the invention provides a method of solving the structure of a crystalline form of a protein, comprising: using at least a portion of the structure co-ordinates of Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) to solve the structure of the crystalline form of a test protein, wherein said test protein has significant amino acid sequence homology to any functional domain of β2AR; and transmitting or storing data descriptive of the structure of said test protein.

In another embodiment, the invention provides a method of identifying from a set of candidate compound three-dimensional models a compound that binds to a ligand binding site of a GPCR or β2AR protein comprising: receiving a three-dimensional model of a ligand binding site on said GPCR or β2AR protein, wherein said three-dimensional model of said ligand binding site comprises atomic co-ordinates for a plurality of ligand binding residues; determining, for each candidate compound of the set of candidate compound three-dimensional models, a plurality of distance and angle values indicating distances and angles between atomic co-ordinates of said candidate compound of the set of candidate compound three-dimensional models and said ligand binding site comprising atomic coordinates of said ligand-binding residues; determining, for each of the set of candidate compound three-dimensional models, a binding strength value based on the plurality of distance and angle values determined for the candidate compound three-dimensional model, wherein the binding strength value indicates the stability of a complex formed by said human GPCR or β2AR protein and a compound represented by the compound three-dimensional model; storing a set of results indicating whether each candidate compound binds to the three-dimensional model based on the binding strength values; searching a database of small organic molecules for compounds exhibiting shape, chemistry, or electrostatic similarity with the candidate compounds indicated to bind to the three-dimensional model based on the binding strength values; and identifying the set of small organic molecules exhibiting shape, chemistry, or electrostatic similarity with the candidate compounds indicated to bind to the three-dimensional model based on binding strength values as likely to also bind to the GPCR or β2AR.the database of small organic molecules is the available chemicals database. In a related embodiment, the shape, chemistry or electrostatic similarity is determined using a program selected from the group consisting of BROOD (openeye), EON (openeye), ROCS (openeye), ISIS Base, and SciFinder.

In another embodiment, the invention provides a method of identifying a ligand that binds to a membrane protein comprising: preparing a lipid meso phase, wherein said lipid meso phase composition comprises (1) a host lipid; (2) said membrane protein; (3) a lipid additive selected from the group consisting of consisting of a sterol, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin, wherein said lipid additive is 1 to 50% w/w in a lipid host; subjecting said lipid meso phase to humidity and temperature conditions to grow crystals of said membrane protein; contacting said membrane protein crystals with diffusible ligands or a mixture of diffusible ligands; determining the three-dimensional structure of said diffusible ligand contacted membrane protein crystals by X-ray crystallography to obtain an electron density map; and identifying bound ligands by inspection of the electron density map. In a related embodiment, the ligands are substantially insoluble in water.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1. Crystals of β2AR-T4L obtained in 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid. FIG. 1a shows β2AR-T4L crystals in the crystallization mixture drop (upper left) and in the loop. FIG. 1b shows crystals in the “sponge” phase.

FIG. 2. Before (top) and after (bottom) images of a lipidic cubic phase crystal harvested directly from a well in a previously sealed glass sandwich plate, according to the method described herein (see, e.g., Example 1).

FIG. 3. Diffraction pattern (2.8 {acute over (Å)} resolution) from β2AR-T4L crystals grown in lipid cubic phase. The crystal size was approximately 25×5×5 {acute over (Å)}; space group C2 (a=106.8 {acute over (Å)}, b=169.5 {acute over (Å)}, c=40.5 {acute over (Å)}; β=105.3°, α=γ=90°. Beam diameter was 10 μm, exposure 10 s, oscillation: 1°.

FIG. 4. Gallery of crystals of various GPCRs obtained using LCP/cholesterol mixtures and in combination with a variety of ligands. The top panel corresponds to non-optimized initial hits, whereas the bottom panel shows diffraction quality for optimized crystals. From left to right, β2AR-T4L (bound to carazolol), diffracted to 2.4 Å resolution; β2AR(E122W)T4L (bound to carazolol), diffracted to 3.5 Å resolution; β2AR(E122W)T4L (bound to alprenolol), diffracted to 3.5 Å resolution; β2AR(E122W)T4L (bound to timolol), diffracted to 2.8 Å resolution; β2AR(E122W) (bound to carazolol); β2AR(E122W)T4L (bound to clenbuterol), diffracted to 6 Å, anisotropic; human A2A adenosine receptor-T4L (bound to ZM241385), diffracted to 2.6 Å resolution.

FIG. 5. A. β2AR-T4L Crystals grown from bicelle conditions. B. Diffraction image from bicelle grown microcrystals of β2AR-T4L recorded using 10 μm minibeam on 231D-B beamline at APS. Black circle is drawn at resolution 3.5 Å.

FIG. 6. A. Microcrystals of β2AR-T4L grown in lipidic mesophase. B. Diffraction image from lipidic cubic phase grown microcrystals of β2AR-T4L recorded using a 10 μm minibeam on 231D-B beamline at APS. The white circle is drawn at resolution 2.2 Å.

FIG. 7. Detailed representation of the carazolol binding site in β2AR-T4L. FIGS. 7A, B, and C are representations of the electron density of the ligand binding site at three different orientations. Residues are labeled by their Ballesteros-Weinstein numbers as superscripts. Electron density is contoured at 16 from a 2 Fo-Fc difference map. Both B and C are generated by rotating the field of view 90° about the y-axis clockwise and counterclockwise respectively.

FIG. 8. Electron density of: A. Cholesterol molecules shown with a Fo-Fe electron density contoured at 2σ omitting the lipid from phase calculation, palmitic acid is also shown. B. Helix-kinked region with 2Fo-Fc electron density contoured at 1.5σ.

FIG. 9. Overall fold of the β2AR-T4L fusion with its predicted orientation in the plasma membrane and key intramolecular interactions. A. Stereoview of the overall fold of β2AR-T4L. The receptor and T4L are colored gray and green, respectively. Carazolol is colored blue and the lipid molecules bound to the receptor are colored yellow. B. The receptor is aligned to a rhodopsin model that was positioned in a lipid membrane (boundaries indicated by horizontal black lines) as found in the orientations of proteins in membranes (OPM) database (M. A. Lomize et al., Bioinformatics 22, 623 (2006)). T4L is fused internally into the third intracellular loop of β2AR and maintains minimal intramolecular packing interactions by tilting away from the receptor. C. Specific intramolecular interactions between β2AR and T4L are represented.

FIG. 10. Crystal packing interactions in the lipidic mesophase crystallized β2AR-T4L. A. There are four main contact areas, two of which are mediated by T4L in the plane of the membrane with itself through a two-fold symmetry axis and translation. The third interaction is normal to the membrane plane between T4L and lumen exposed loops of β2AR. The fourth interaction is generated by the two-fold symmetry axis, packing one receptor to receptor in the plane of the membrane. B. The receptor crystal packing interface is composed mainly of lipids with two cholesterol molecules and two palmitic acid molecules forming the majority of the interactions. A network of ionic charge interactions exists on the cytoplasmic end of the interface forming the only inter-receptor protein contacts. C. Comparison between β2AR-T4L and rhodopsin (PDB ID Code 2135) parallel receptor association interface. Helices I (blue) and VIII (magenta) are highlighted in both structures. Only one monomer is shown for each receptor representation along with helices I′ and VIII′ only from the opposing symmetry related molecule. The rhodopsin interface is twisted significantly relative to β2AR-T4L resulting in a significant offset from the parallel orientation required for a physiological dimer interface. β2AR-T4L associated monomers are in a highly parallel orientation.

FIG. 11. Surface representation of β2AR colored by calculated charge from red (−10 kbT/ec) to blue (+10 kbT/ec) using a dielectric constant of 70. A. Three main areas of interest are indicated. The binding site cleft is negatively charged as is a groove between helices III, IV and V. The third region is an overall positive charge in the region of the ionic lock and DRY motif on the cytoplasmic face. The overall result is a highly polarized molecule that may utilize its negative charge to facilitate binding of catecholamine ligands. The presence of a negative charge in the groove between helices III, IV and V is unexpected as it is in the middle of the lipid membrane. This charge may be partially derived from the presence of an unpaired glutamate at position 1223.41. The effective charge in this region is likely greater than shown here due to its location in the low dielectric environment of the lipid membrane. B. View rotated 90° from A. Showing both the negatively charged binding site cleft (top) and positively charged cytoplasmic face (bottom). Poisson-Boltzmann electrostatics were calculated using the program APBS (Baker et al., Proc Natl Acad Sci U S A, 98, 10037 (2001)) as implemented in Pymol (The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org). Pymol was used exclusively in the preparation of all figures.

FIG. 12. Comparison of the extracellular sides of β2AR-T4L and rhodopsin. A. The N-terminus is missing from the experimental density in the β2AR-T4L structure and is not shown. ECL2 is shown in green and contains a short α-helix and two disulfide bonds (yellow). The intraloop disulfide bond constrains the tip of ECL2 which interacts with ECL1. The second disulfide bond links ECL2 with helix III. There is one interaction between ECL2 and carazolol (blue) through Phe1935.32. The entire loop is held out of the ligand binding site by a combination of the rigid helical segment and the two disulfide bonds. B. In contrast, ECL2 (green) in rhodopsin assumes a lower position in the structure that occludes direct access to the retinal-binding site and forms a small β-sheet in combination with the N-terminal region (magenta) directly above the bound retinal (pink).

FIG. 13. Ligand binding characterization and comparison to rhodopsin. A. A view looking down on the plane of the membrane from the extracellular surface showing a detailed representation of the carazolol binding site in β2AR-T4L. Carazolol is shown as sticks with carbon atoms colored yellow. β2AR-T4L residues contributing to carazolol binding are shown in green and labeled. Electron density is contoured at 5σ from an Fo-Fc omit map calculated without the contribution of carazolol. B. Binding orientation comparison between 11-cis-retinal in rhodopsin and carazolol in β2AR-T4L. Van der Waals' surfaces for carazolol and retinal are represented as dots to accentuate the close packing interactions. Retinal in the all-cis conformation (pink), binds deep in the active site of rhodopsin as compared to carazolol (blue), packing its β-ionone ring between Tyr2686.51 and Phe2128.47 (cyan), blocking movement of Trp2656.48 (magenta) into the space. The β-ionone ring of trans-retinal in activated rhodopsin would not block Trp2656.48 from rotating into the space allowing a rotameric shift into its proposed active form. C. There are four residues involved in the toggle switch mechanism of β2AR-T4L as shown. Phe2906.52 (magenta) is sandwiched between Phe2085.47 (tan) and Phe2896.51 (tan) forming a ring-face aromatic interaction. Like rhodopsin, an activation step is thought to occur by a rotameric change of Trp2866.48 (magenta) which would displace Phe2906.52. Carazolol is shown to interact extensively with the sandwich motif as shown: however, few interactions are seen with Trp2866.48. The 6.52 position in β2AR-T4L is occupied by Phe2906.82 as opposed to Ala2696.82 in rhodopsin where the β-ionone ring replaces an aromatic protein side chain in forming the sandwich interactions. The aromatic character of the sandwich is otherwise maintained by Phe2896.81 and Phe2088.47 in β2AR-T4L.

FIG. 14. Comparison of β2AR-T4L helical orientations with rhodopsin (PDB ID Code 1U19). A. β2AR-T4L is rendered as a ribbon trace colored with a blue to red spectrum corresponding to observed distances between Cα positions in the two structures (RMSD 2.7 Å between all residues in the transmembrane region). Helix II shows very little movement, whereas the entire lengths of helices III, IV, V shift significantly. Helix VIII and loops were not included in the comparison and are colored in tan. B. Movements of helices I and V of rhodopsin (grey) are shown relative to β2AR-T4L. C. Movements of helices III, IV and VI. D. Ligand binding site representation. Carazolol is shown with yellow carbons. Entire helices are assigned a single designation based on their divergence from the rhodopsin position in the area of the ligand binding site as shown. Helix I is highly divergent, Helices II and VI are similar to rhodopsin. Helices IV and VII are moderately constant. Helices III and V are moderately divergent.

FIG. 15. Affinity curves for adrenergic ligands binding to β2AR-T4L and wildtype β2AR. Saturation curves for the antagonist [3H]DHA is shown at left, next to competition binding curves for the natural ligand (−)-Epinephrine and the high-affinity synthetic agonist Formoterol. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described above.

FIG. 16. Comparison of the proteolytic stability between the wild-type β2AR and β2AR-T4L in a limited trypsin proteolysis assay. TPCK-trypsin was added to carazolol-bound, purified, dodecylmaltoside-solubilized receptor at a 1:1000 ratio (wt:wt), and samples were analyzed by SDS-PAGE. Intact β2AR-T4L (56.7 kD) and FLAG-tagged wild-type β2AR (47.4 kD) migrate similarly as ˜55 kD bands. Markers are Biorad low-range SDS-PAGE protein standards.

FIG. 17. Stability comparison of unliganded β2AR365 and β2AR-T4L. For dodecylmaltoside-solubilized receptor preparations, maintenance of the ability to specifically bind [3H]DHA after incubation at 37° C. is taken as a measure of stability.

FIG. 18. Superimposed Cα traces of the receptor component of β2AR-T4L (in blue) and β2AR365 (in yellow). Common modeled transmembrane helix regions 41-58, 67-87, 108-137, 147-164, 204-230, 267-291, 312-326, 332-339 were used in the superposition by the program Lsqkab (The CCP4 Suite, Acta Crystallogr D Biol Crystallogr 50, 760 (1994)) (RMSD=0.8 Å).

FIG. 19. Carazolol dissociation from β2AR365. Dodecylmaltoside-solubilized carazolol-bound receptor (at 50 μM) was dialyzed in a large volume of buffer containing 300 micromolar alprenonol as a competing ligand, and aliquots were removed from the dialysis cassette at different time points. Remaining bound carazolol was measured (in a relative sense) by collecting fluorescence emission with excitation at 330 nm and emission from 335 to 400 nm. For each carazolol fluorescence measurement, data was normalized for the protein concentration in the dialysis cassette (measured with the Bio-Rad Protein DC kit). The Y-axis represents carazolol fluorescence emission Intensity (in cps) at 341 nm. The exponential decay of carazolol concentration in the receptor dialysis cassette was fit using Graphpad Prism software, giving a half-life of 30.4 hrs.

FIG. 20. Comparison of β1 and β2AR sequences. After aligning the β1 and β2AR sequences, positions that have different amino acids between the two receptors were mapped onto the high-resolution structure of β2ART4L (shown as red sticks). The carazolol ligand is shown as green sticks (with nitrogens in blue and oxygens in red). Highlighted residues Ala852.56, Ala922.63 and Tyr3087.35 are homologous to amino acids Leu1102.56, Thr1172.63 and Phe3597.35 of the β1AR, which were shown to be primarily responsible for its selectivity over β2AR for the compound RO363 (Sugimoto et al., J Pharmacol Exp Ther 301, 51 (2002)). In the β2AR-T4L structure, only Tyr3087.35 faces the ligand, while Ala852.56 lies at the interface between helices II and III. Of all the divergent amino acids, only Tyr3087.35 is found within 4 Å of any atom of carazolol.

FIG. 21. Design and optimization of the β2AR-T4L fusion protein A. The sequence of the region of the β2AR targeted for insertion of a crystallizable domain is shown (SEQ ID NO:2), and the positions of the junctions between the receptor and T4L (in red) for various constructs are indicated. The sequences that were initially replaced or removed are faded. Red lines are shown after every tenth residue. Peptide ‘LNKYADWT’ disclosed as SEQ ID NO: 3 B. Immunofluorescence images of HEK293 cells expressing selected fusion constructs. Panels on the left shows M1 anti-FLAG signal corresponding to antibody bound to the N-terminus of the receptor. Panels on the right show the same signal merged with blue emission from DAPI (nuclear staining for all cells). Plasma membrane staining is observed in the positive control, D3 and D1, while C3 and D5 are retained in the endoplasmic reticulum.

FIG. 22. Functional characterization of β2AR-T4L. A. Affinity competition curves for adrenergic ligands binding to β2AR-T4L and wild-type β2AR. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described in the methods section of Example 4. B. β2AR-T4L is still able to undergo ligand-induced conformational changes. Bimane fluorescence spectra (excitation at 350 nm) of detergent-solubilized β2AR-T4L and wild-type β2AR truncated at 365, labeled under conditions that selectively modify Cys2656.27 (see methods section of Example 4), were measured after incubating unliganded receptor with compounds for 15 min at room temperature. The cartoon illustrates that the observed changes in fluorescence can be interpreted as a movement of the bimane probe from a more buried, hydrophobic environment to a more polar, solvent-exposed position.

FIG. 23. A. Side-by-side comparison of the crystal structures of the β2AR-T4L fusion protein and the complex between β2AR365 and a Fab fragment. The receptor component of the fusion protein is shown as a blue cartoon (with modeled carazolol as red spheres), while the receptor bound to Fab5 is in yellow. B. Differences in the environment surrounding Phe2646.26 (shown as spheres) for the two proteins. C. The analogous interactions to the “ionic lock” between the E(D)RY motif and Glu2476.30 seen in rhodopsin (right panel, darkened) are broken in both structures of the β2AR (left panel). Pymol (W. L. DeLano, The PyMOL Molecular Graphics System (2002) on the World Wide Web http://www.pymol.org) was used for the preparation of all figures.

FIG. 24. Schematic representation of the interactions between β2AR-T4L and carazolol at the ligand binding pocket. Residues shown have at least one atom within 4 Å of the ligand in the 2.4 Å resolution crystal structure.

FIG. 25. The ligand binding pocket of β2AR-T4L with carazolol bound. A. Residues within 4 Å of the ligand are shown as sticks, with the exception of A200, N293, F289, and Y308. Residues that form polar contacts with the ligand (distance cutoff 3.5 Å) are in green, other residues are gray (in all panels, oxygens are colored red and nitrogens are blue). B. Same as panel A, except that the ligand is oriented with its amine facing out of the page. W109 is not shown. C. Packing interactions between carazolol and all residues within 5 Å of the ligand. View is from the extracellular side of the membrane. Carazolol is shown as yellow spheres, receptor residues are shown as sticks within van der Waals dot surfaces. D. Model of (−)-isoproterenol (magenta sticks) in the ligand binding pocket observed in the crystal structure. A model of the agonist with optimal bond lengths and angles was obtained from the PRODRG server (Schuettelkopf, et al., Acta Crystallogr D Biol Crystallogr D60, 1355 (2004)), and the dihedral angles were adjusted to the values observed in the homologous atoms of bound carazolol (16-22 in FIG. 24). The one remaining unaccounted dihedral in (−)-isoproterenol was adjusted in order to place the catechol ring in the same plane as the C16-C15-O14 plane in carazolol. Residues known to specifically interact with agonists are shown as green sticks.

FIG. 26. Packing interactions in the β2AR that are likely to be modulated during the activation process. A. On the left, residues previously demonstrated to be CAMs (Rasmussen et al., Mol Pharmacol 56, 175 (1999); Tao, et. al., Mol Endocrinol 14, 1272 (2000); Jensen et al., J Biol Chem 276, 9279 (2001); Shi et al., J Biol Chem 277, 40989 (2002); Zuscik, et. al., (1998)) or UCMs (Strader et al., Proc Natl Acad Sci U S A 84, 4384 (1987); Chung, et al., J Biol Chem 263, 4052 (1988); Moro, et. al., J Biol Chem 269, 6651 (1994); Green, et. al., J Biol Chem 268, 23116 (1993); Gabilondo et al., Proc Natl Acad Sci U S A 94, 12285 (1997)) are shown as van der Waals spheres mapped onto a backbone cartoon of the β2AR-T4L structure. On the right, residues that are found within 4A of the CAMs Leu1243.43 and Leu2726.34 are shown as yellow spheres or dot surfaces. A vertical cross-section through the structure illustrates that these surrounding residues connect the CAMs on helices III and VI with the UCMs on helix VII through packing interactions. B. In both β2AR-T4L (blue) and rhodopsin (purple), a network of ordered water molecules is found at the interface between the transmembrane helices at their cytoplasmic ends. C. Network of hydrogen bonding interactions between water molecules and β2AR-T4L residues (sidechains as blue sticks), notably the UCMs on helix VII (orange cartoon).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides methods and compositions for generating crystal structures of membrane proteins that diffract to resolutions as low as 2 to 3 {acute over (Å)}ngstroms. In one embodiment, the methods involve the crystallization of proteins in a lipidic cubic phase, wherein the host lipid comprises an additive, e.g., a sterol, such as cholesterol. The invention also provides the crystallized membrane proteins themselves, wherein the crystallized membrane proteins include GPCRs or modified GPCRs. The crystallized proteins can also include bound ligands, natural agonists, antagonists, and/or allosteric effectors. The invention additionally provides methods of using the 3-dimensional structures of the proteins (obtained from the crystals) to screen for novel ligands, drugs, and other useful molecules that affect the conformation and/or activity of the proteins in vitro or in vivo.

More specifically, the invention provides particular crystal forms of GPCRs diffracting to high resolutions. GPCRs have been grouped into five classes (Fredriksson, et al., Mol Pharmacol 63, 1256 (2003)) based on sequence conservation, with class A GPCRs, including β2AR, being the largest and most studied. β2AR agonists are used in the treatment of asthma and preterm labor (DeLano, The PyMOL Molecular Graphics System (2002) on World Wide Web at pymol.org). The crystal forms provided by the invention include several diffraction-quality class A GPCR crystals, including crystals comprising β2AR and crystals comprising the human adenosine A2A receptor.

The invention provides a three-dimensional structure of a human β2AR protein comprising a T4-lysozyme (T4L) in place of the third intracellular loop (“β2AR-T4L”) that has been solved in the presence of carazolol (2-propanol, 1-(9H-carbazol-4-yloxy)-3-[(1-methylethyl)amino] at 2.4 Å resolution. Additional class A GPCR structures make it possible to correlate sequence differences between GPCRs, e.g., between rhodopsin and β2AR, with empirically determined structural differences and extrapolate to other class A GPCRs. Highlighting interactions that constrain class A receptors into each of the two observed states allows a more comprehensive analysis of structural divergence and, therefore, more accurate models. Furthermore, GPCR structures provide an alternative signaling state on which to base homology models that will be more relevant for virtual ligand screening and structure-based drug design (Bissantz, et. al, Proteins 50, 5 (2003); Gouldson et al., Proteins 56, 67 (2004)).

DEFINITIONS

Terms used in the claims and specification are defined as set forth below unless otherwise specified. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

As used herein, the term “binding site” or “binding pocket” refers to a region of a protein that binds or interacts with a particular compound.

As used herein, the terms “binding” or “interaction” refers to a condition of proximity between a chemical entity, compound, or portions thereof, with another chemical entity, compound or portion thereof. The association or interaction can be non-covalent—wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions—or it can be covalent.

As used herein, the term “residue” refers to an amino acid that is joined to another by a peptide bond. Residue is referred to herein to describe both an amino acid and its position in a polypeptide sequence.

As used herein, the term “surface residue” refers to a residue located on a surface of a polypeptide. In contrast, a buried residue is a residue that is not located on the surface of a polypeptide. A surface residue usually includes a hydrophilic side chain. Operationally, a surface residue can be identified computationally from a structural model of a polypeptide as a residue that contacts a sphere of hydration rolled over the surface of the molecular structure. A surface residue also can be identified experimentally through the use of deuterium exchange studies, or accessibility to various labeling reagents such as, e.g., hydrophilic alkylating agents.

As used herein, the term “polypeptide” refers to a single linear chain of 2 or more amino acids. A protein is an example of a polypeptide.

As used herein, the term “homolog” refers to a gene related to a second gene by descent from a common ancestral DNA sequence. The term, homolog, can apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication.

As used herein, the term “conservation” refers to a high degree of similarity in the primary or secondary structure of molecules between homologs. This similarity is thought to confer functional importance to a conserved region of the molecule. In reference to an individual residue or amino acid, conservation is used to refer to a computed likelihood of substitution or deletion based on comparison with homologous molecules.

As used herein, the term “distance matrix” refers to the method used to present the results of the calculation of an optimal pairwise alignment score. The matrix field (i,j) is the score assigned to the optimal alignment between two residues (up to a total of i by j residues) from the input sequences. Each entry is calculated from the top-left neighboring entries by way of a recursive equation.

As used herein, the term “substitution matrix” refers to a matrix that defines scores for amino acid substitutions, reflecting the similarity of physicochemical properties, and observed substitution frequencies. These matrices are the foundation of statistical techniques for finding alignments.

As used herein, the term “pharmacophore” refers to an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target structure and to trigger or block a biological response. A pharmacophore can be used to design one or more candidate compounds that comprise all or most of the ensemble of steric and electronic features present in the pharmacophore and that are expected to bind to a site and trigger or block a biological response.

As used herein, the term “G-protein coupled receptor” (or “GPCR”) refers to a member of a family of heterotrimeric guanine-nucleotide binding protein (“G-protein”) coupled receptors (Pierce, et al., Nat. Rev. Mol. Cell. Biol. 3:630 (2002)). GPCRs share a common structural signature of seven membrane-spanning helices with an extra-cellular N terminus and an intracellular C terminus. The family has been grouped into at least five classes (designated A, B, C, D, E, etc.; see, e.g., Fredriksson, et al., Mol Pharmacol 63, 1256 (2003)) based on sequence conservation. When used without a descriptive limitation, the term “a G-protein couple receptor” includes GPCRs with native amino acid sequences as well as genetically engineered or otherwise mutated GPCR proteins. Mutated GPCR proteins include those comprising point mutations, truncations, inserted sequences or other chemical modifications, while retaining ligand binding activity. One example of a GPCR referred to herein that comprises a point mutation is β2ARE122W. An example of a GPCR referred to herein that comprises an inserted T4 lysozyme sequence is the human A2a adenosine receptor-T4L.

Adrenergic receptors in the class A or amine group are some of the most thoroughly investigated GPCRs (Kobilka, Annu Rev Neurosci 15, 87 (1992); Caron, et al., Recent Prog Horm Res 48, 277 (1993); Strosberg, Protein Sci 2, 1198 (1993); Hein, et al., Trends Cardiovasc Med 7, 137 (1997); Rohrer, J Mol Med 76, 764 (1998); Xiang, et al., Adrenergic Receptors, 267 (2006)), and are composed of two main subfamilies, α and β, which differ in tissue localization and ligand specificity, as well as in G protein coupling and downstream effector mechanisms (Milligan, et al., Biochem Pharmacol 48, 1059 (1994)). Some representative class A receptors include the human A2A adenosine receptor and the beta-2 adrenergic receptor. The term “beta-2 adrenergic receptor” (or “β2AR” or “β2AR”) refers to a class A GPCR that responds to diffusible hormones and neurotransmitters and resides predominantly in smooth muscles throughout the body. When used without a descriptive limitation, the term “β2AR” includes β2ARs with native amino acid sequences as well as genetically engineered or otherwise mutated β2AR proteins. Mutated β2AR proteins include those comprising point mutations, truncations, inserted sequences or other chemical modifications, while retaining ligand binding activity. One example of a β2AR referred to herein that comprises a point mutation is β2ARE122W. An example of a β2AR referred to herein that comprises an inserted T4 lysozyme sequence is the human adenosine receptor β2ARE122W-T4L.

The term “diffracts to a resolution of xx-yy Angstroms” means that diffraction data exceeding a predetermined signal to noise ratio can be obtained within the stated resolution range. In some embodiments, that diffraction data can be obtained using synchrotron radiation. Also, in some embodiments, that diffraction data can be obtained following freezing of the crystal in liquid nitrogen.

As used herein, the term “atomic co-ordinates” refers to a set of three-dimensional co-ordinates for atoms within a molecular structure. In one embodiment, atomic-co-ordinates are obtained using X-ray crystallography according to methods well-known to those of ordinarily skill in the art of biophysics. Briefly described, X-ray diffraction patterns can be obtained by diffracting X-rays off a crystal. The diffraction data are used to calculate an electron density map of the unit cell comprising the crystal; said maps are used to establish the positions of the atoms (i.e., the atomic co-ordinates) within the unit cell. Those of skill in the art understand that a set of structure co-ordinates determined by X-ray crystallography contains standard errors. In other embodiments, atomic co-ordinates can be obtained using other experimental biophysical structure determination methods that can include electron diffraction (also known as electron crystallography) and nuclear magnetic resonance (NMR) methods. In yet other embodiments, atomic co-ordinates can be obtained using molecular modeling tools which can be based on one or more of ab initio protein folding algorithms, energy minimization, and homology-based modeling. These techniques are well known to persons of ordinary skill in the biophysical and bioinformatic arts, and are described in greater detail below.

Atomic co-ordinates for binding pockets, such as, e.g., the ligand binding pocket of β2AR, and/or other agonist/antagonist binding sites of the present invention are intended to encompass those co-ordinates set out in the .pdb file (Appendix I; SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) incorporated into this specification, as well as co-ordinates that are substantially equivalent. Substantially equivalent co-ordinates are those that can be related to a reference set of co-ordinates by transformation reflecting differences in the choice of origin or inter-axis angels for one or more axes used to define the coordinate system. Operationally, co-ordinates are “substantially equivalent” when the structures represented by those co-ordinates can be superimposed in a manner such that root mean square deviations (RMSD) of atomic positions for the structures differs by less than a predetermined threshold. In some embodiments that threshold is less than about 5 Angstroms, or less than about 4 Angstroms, or less than about 3 Angstroms, or less than about 2 Angstroms, or less than about 1 Angstrom, or less than about 0.9 Angstrom, or less than about 0.8 Angstrom, or less than about 0.7 Angstrom, or less than about 0.6 Angstrom, or less than about 0.5 Angstrom, or less than about 0.4 Angstrom, or less than about 0.3 Angstrom. Preferably, co-ordinates are considered “substantially equivalent” when the RMSD is less than about 1 Angstrom. Methods for structure superpositioning and RMSD calculations are well known to those of ordinary skill in the art, and can be carried out using programs such as, e.g., the programs listed in Table 5 below.

Structural similarity can be inferred from, e.g., sequence similarity, which can be determined by one of ordinary skill through visual inspection and comparison of the sequences, or through the use of well-known alignment software programs such as CLUSTAL (Wilbur et al., J. Proc. Natl. Acad. Sci. USA, 80, 726 730 (1983)) or CLUSTALW (Thompson et al., Nucleic Acids Research, 22:4673 4680 (1994)) or BLAST®. (Altschul et al., J Mol. Biol., October 5; 215(3):403 10 (1990)), a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. CLUSTAL W is available at the EMBL-EBI website (http://www.ebi.ac.uk/clustalw/); BLAST is available from the National Center for Biotechnology website (http://www.ncbi.nlm.nih.gov/BLAST/). A residue within a first protein or nucleic acid sequence corresponds to a residue within a second protein or nucleic acid sequence if the two residues occupy the same position when the first and second sequences are aligned.

The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence co-ordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI web-site).

The term “sterol” refers to a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring. See Fahy E. Subramaniam S et al., “A comprehensive classification system for lipids,” J. Lipid Res. 46 (5):839-861 (2005)). Sterols are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. Sterols can include, e.g., cholesterol or cholesteryl hemisuccinate (“CHS”).

The term “atomic co-ordinates for residues” refers to co-ordinates for all atoms associated with a residue, or for some of the atoms such as, e.g., side chain atoms.

The term “atomic co-ordinates of a candidate compound” refers to co-ordinates for all atoms comprising the compound or a subset of atoms comprising the compound.

The term “characterizing a binding interaction” refers to characterizing any observable property of a first molecule and determining an whether there is a change in that observable property after contacting the first molecule with a second molecule under conditions in which said first and second molecules can potentially bind.

The term “antagonist” refers to molecules that bind to and block the active site of a protein, but do not affect the equilibrium between inactive and active states. In contrast, an “agonist” is a ligand that shifts the equilibrium to an active receptor state. An “inverse agonist” is a ligand that acts to reduce the basal activity of a receptor through interactions that shift the equilibrium to more of an inactive state.

Ballesteros-Weinstein numbering appears in the text and Figures as superscripts to the protein numbering. Within each helix is a single most conserved residue among the class A GPCRs. This residue is designated X.50, where x is the number of the transmembrane helix. All other residues on that helix are numbered relative to this conserved position.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Method for Producing Diffraction Quality Crystals of Membrane Proteins

In one aspect, the present invention discloses a modified lipidic cubic mesophase method for crystallizing proteins (see, e.g., Cherezov et al., Biophysical J., v. 83, 3393-3407 (2002)). The novel method described herein yields diffraction quality crystals of membrane proteins and is particularly useful for generating crystals of G-protein coupled receptor proteins (“GPCRs”). The method has now been successfully applied to diverse members of this important family of proteins, yielding crystals that diffract to resolutions in the 2.5 {acute over (Å)} range. Among other advantages, this method allows diffraction-quality crystals of membrane proteins to be generated in the absence of any stabilizing antibodies bound to the protein in the crystal.

The LCP/sterol crystallization method described herein includes a step of mixing a solution containing the protein of interest with a host lipid or a host lipid mixture that includes a lipid additive. Given the teaching provided herein, one skilled in the art will recognize that a variety of host lipids may suffice for the generation of a cubic mesophase, e.g., hydrated monounsaturated monoacylglycerols such as monoolein, monopalmitolein, and/or monovacennin. The host lipid 1-monoolein is a preferred host lipid for certain applications of the method. In embodiments utilizing a lipid mixture, a lipid additive that is distinct from the host lipid is included, e.g., monounsaturated monoacylglycerols or other hydrophobic molecules known to interact with membranes or membrane-associated proteins such as, 2-monoolein, phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid, dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoyl phosphatidylcholine (“DOPC”), Asolectin, or a sterol (e.g., cholesterol, ergosterol, etc.). An example of a lipid mixture for GPCR crystallization is one comprising cholesterol as lipid additive in a ratio between 1 and 50% w/w relative to the host lipid, more preferably between 5 and 20%, and even more preferably between 8 and 12%. The protein mixture may include ligands of physiological interest and/or ligands that stabilize the protein. In the case of GPCRs, the ligands may include various agonists and antagonists known to the artisan, including well-known agonists such as carazolol (an inverse agonist), timolol, and other molecules including, without limitation, Examples of ligands, include but are not limited to carazolol, light and olfactory stimulatory molecules; adenosine, bombesin, bradykinin, endothelin, y-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), glucagon, acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, C5a anaphylatoxin, follicle stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropin releasing hormone (TRH), and oxytocin).

A typical concentration of protein in the protein mixture is 25-75 mgs/ml but this concentration may vary according to protein identity and purification methods. As will be recognized by the skilled artisan, the concentration must be high enough to result in a degree of insolubility sufficient for nucleation to occur after a precipitation solution is combined with the protein-laden lipid solution; on the other hand, concentrations of protein that are too high may prevent the orderly growth of high-quality crystals.

The lipid mixture is preferably combined with the protein mixture and homogenized, e.g., using a syringe mixer, spontaneously yielding a homogenous cubic phase. Typically, the lipid mixture is added to the protein solution at a ratio of 1:1, 3:2, 4:2 w/w lipid:protein, but this ratio may be varied by the skilled artisan as desired, depending on various parameters, e.g., the concentration of protein in the protein mixture. The protein-laden lipidic cubic phase preparation thus obtained is then combined with precipitation solution (also referred to as crystallization solution) on or in an appropriate surface or container, e.g., a glass sandwich plate with wells where the mixed solutions can incubate while crystallization occurs. A typical volume of the protein-laden lipidic cubic phase used in the method is between 10 and 100 nL, with 40 to 60 nL preferred in certain embodiments. A typical volume of precipitation solution is 20 to 100 times greater, e.g., for a 20 nL volume of protein-laden lipidic cubic phase, approximately 1 μL of precipitation solution would be added to initiate crystallization.

The precipitation solution used in the crystallization method is an appropriately buffered solution (i.e., buffered to approximate the physiological conditions of the native protein) comprising polyethylene glycol, a salt, and optionally a small soluble molecule such as an alcohol.

With respect to the polyethylene glycol in the precipitation solution, useful PEG molecules include PEG 300, PEG 400, PEG 550, PEG 550mme, PEG 1000, and PEG 1500, as well as other PEG molecules with average molecular weights less than 2000. In certain embodiments, larger average molecular weight PEG molecules (up to 20,000) or modified PEG molecules may be preferred. In some embodiments, the PEG or modified PEG has an average molecular weight of 400. Examples of modified PEG include but are not limited to PEG laurate, PEG dilaurate, PEG oleate, PEG dioleate, PEG stearate, PEG distearate, PEG glyceryl trioleate, PEG glyceryl laurate, PEG glyceryl stearate, PEG glyceryl oleate, PEG palm kernel oil, PEG hydrogenated castor oil, PEG castor oil, PEG corn oil, PEG caprate/caprylate glycerides, PEG caprate/caprylate glycerides, PEG cholesterol, PEG phyto sterol, PEG soya sterol, PEG trioleate, PEG sorbitan oleate, PEG sorbitan laurate, PEG succinate, PEG nonyl phenol series, PEG octyl phenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHS Ester and methoxypoly(ethylene glycol) (mPEG).

PEG may be present in the crystallization solution in concentrations between 10-60% v/v, and most typically between 20-40% v/v. The preferred concentration will vary depending on the average molecular weight of PEG utilized, i.e., 10-60% v/v of PEG will be preferred for PEG ≦1000 whereas 10-30% w/v will be preferred for PEG >1000 (larger average molecular weight PEG formulations are described in % w/v rather than % v/v).

With respect to the salt used in the method, an optimum cation can usually be found for a given crystal. Both sodium and lithium sulfate have proven useful for obtaining high resolution proteins of GPCRs. Again, the concentrations may be varied up to 1M, with lower concentrations of approximately 50-200 mM typically preferred. Other organic salts, e.g., citrate, malonate, tartrate, formate and acetate, may also be screened for their effects on crystal formation. In certain embodiments, the precipitation solution additionally comprises a small organic molecule such as an alcohol, a diol or a triol, e.g., a hexanediol, a butanediol, or derivative thereof. These molecules may be present in the precipitation solution in various concentrations, as appropriate, but typically in the range of 1-20% v/v, more typically in the 5-10% v/v range. In certain embodiments, preferred combinations of lipid additives (in the protein-laden lipidic cubic phase mixture) and small molecules (in the precipitation solution) yield optimal results. Examples of such combinations include 1,4-butanediol in combination with DOPE or cholesterol, and 2,6-hexanediol in combination with cholesterol.

In optimizing the conditions from micro-crystals to larger crystals for a given system (e.g., a protein/ligand system), the choice and concentration of a specific sterol(s) and specific lipid(s), as well the pH, buffer system, salt, and salt concentration may be varied, as in other types of crystallization formats. As noted above, small organic additives, especially alcohols and diols such as 1,4 butanediol, 1,6 hexanediol, etc., can be particularly useful in generating large diffraction quality crystals. Also, due to the membrane fluidity-altering properties of cholesterol and other sterols, sterol and precipitant concentration should be treated as dependent variables. For example, increasing concentrations of cholesterol in monoolein serve to rigidify the membranes, potentially slowing diffusion of the membrane protein within the lipid matrix. Conversely, increasing concentrations of PEG 400 swell the cubic phase, thereby increasing the lattice parameter of the matrix and speeding diffusion within the lipid. The former scenario would slow the rate of crystallization while the latter would increase the rate. The two effects should therefore be balanced for optimal nucleation and also for optimal growth of large, well-ordered crystals that diffract to a high resolution.

The mixing of the protein-laden lipidic cubic phase solution and the precipitation solution typically occurs at room temperatures. After set-up, the plates containing the mixed crystallization solutions can be monitored as often as desired for the appearance of crystal growth. One skilled in the art will recognize that further optimization of these conditions may be desirable, for example, to maximize the size and number of diffraction quality crystals that are obtained. In making determinations as to the preferred molecules and conditions for crystallization, the skilled artisan may rely on well-known phase diagrams and other previously determined physical constants, in addition to the novel methodology and Examples described herein. For certain lipid mixtures, pre-screening their phase behavior by microscope visualization and/or by X-ray prior to being mixed with the protein solution may facilitate the process of optimization. An in meso crystallization robot and automatic imager combined with multiple 96-well optimization screens can be used to run thousands of trials in a relatively facile manner.

It also possible to achieve additional stabilization of proteins and improve the yield of diffraction-quality crystals using the LCP/sterol method described herein by modification of the protein. For example, an unstable region of the protein may be replaced or stabilized by incorporation of a portion of a stable protein, e.g., a T4 lysozyme, whose structure is previously known but which does not (when fused) significantly affect the biochemical activity of the protein of interest. For example, the ECL2 and ECL3 regions of a β2AR can be stabilized by such modifications, as described herein (Examples 3 and 4). Other modifications include one or more point mutations that do not significantly alter the properties of the protein of interest except to increase its stability and/or tendency to crystallize well. For example, β2AR(E122W) comprises an E122W point mutation and yields crystals with the LPC/sterol method. Analogous residues in other GPCRs could be modified in the same way. One advantage of the LCP method applied to both modified and unmodified proteins, as noted above, is that it allows (but does not preclude) the crystallization of proteins in the absence of heterologous proteins, such as antibodies, that may not be of interest to the crystallographer.

Method of Ligand Screening by Lipidic Cubic Phase Crystallization

Aspects of the lipidic cubic phase crystallization methodology described above can be modified for the purpose of determining low affinity lipid binding sites within integral membrane proteins through co-crystallization trials within a lipidic cubic phase matrix. In this method various lipids of different composition are incorporated at a variety of concentrations into monoolein, wherein the monoolein is solubilized in chloroform or heated to its fluid isotropic phase. Crystal growth is then assessed by visual inspections and diffraction data collected on any crystalline material within the experiment. Because the lipid is low affinity the method requires an environment conducive for free exchange of lipid from annular to non-annular protein binding sites. The presence of interpretable electron density not associated with crystal packing interfaces allows the inference of specific binding sites for a particular lipid within the context of the membrane protein in a membrane environment. Because the binding occurs within a membrane the complicating factor of detergent partitioning is eliminated and thermodynamics of association are more realistic. The method thus allows one to characterize in detail previously inaccessible regions of membrane proteins, as well as describe and exploit binding interactions that might otherwise remain undetected. Furthermore, the technique can be applied to ligand binding studies where the ligand occupies a site on the membrane protein that is juxtaposed to the lipid plane relying on partitioning into the aqueous phase to enable saturation of the site. This limits the exposure of the protein to harsh organic co-solvents and may also find utility for soluble proteins that crystallize within the lipidic cubic phase solvent channels.

By wan of example, existing crystallization conditions for a protein can be utilized as a starting point for screening novel ligands to the β2-adrenergic receptor. In the first instance, cholesterol solubilized in chloroform can be incorporated into chloroform-solubilized monoolein at weight ratio of 10%. After drying and desiccating the mixture, protein at 30-80 mg/mL can incorporated at 2/3 volume ratio and used for crystallization trials. A similar protocol was used for other lipid like molecules, including cholesteryl hemisuccinate and a variety of other cholesterol analogs. In each case protein was incorporated into the resulting mixture and screened for crystallization. Binding of the novel ligand to the receptor is indicated by diffraction quality crystals and ultimately by three-dimensional structural data. By incorporating analogues of cholesterol we are able to map out the binding specificities based on the unique structural features of their respective sterol rings and polar moieties and, if their incorporation led to diffraction quality crystals, the interactions between the protein and cholesterol analogue are determined.

This method of ligand screening is not limited to lipid-like molecules, as we can use the lipidic cubic phase as a host for other highly hydrophobic molecules that act at orthosteric binding sites. One problem with structure based or fragment based design of novel ligands is the hydrophobicity often associated with potential drug leads. This is a problem in aqueous based crystallization schemes because the solubility of the ligand is often less than 1 mM and unless there is a slow off rate from the protein of interest the binding site will be in a ligand depleted state at crystallization conditions which often involve protein concentrations between 0.5 and 1 mM. One can attempt to co-solubilize the hydrophobic ligand in aqueous miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). However, these often interfere with the stability or crystallization properties of the protein and their usefulness is not general. Therefore, this method allows one to incorporate the hydrophobic ligand directly into the lipidic cubic phase where its accessibility to the protein will be limited by the partitioning between the lipid and aqueous phase and/or the accessibility of the binding.

Additional guidance relating to these methods is provided by the working and prophetic examples of protein crystallization presented herein.

The Crystal Structure of Human β2AR Bound to Carazolol and Uses Thereof

G-protein coupled receptors are cell surface receptors that indirectly transduce extracellular signals to downstream effectors, e.g., intracellular signaling proteins, enzymes, or channels. G-protein coupled receptor membrane proteins are grouped into one of at least 6 classes (i.e., A, B, C, D, E, and F). An example of a mammalian G-protein coupled receptor is the β2A receptor, a receptor in the Class A subfamily of GPCRs.

Class A GPCRs function in a variety of physiological processes such as vasodilation, bronchodilation, neurotransmitter signaling, stimulation of endocrine secretions, gut peristalsis, development, mitogenesis, cell proliferation, cell migration, immune system function, and oncogenesis. Accordingly, class A GPCRs can be used as screening targets to identify modulators of these processes which can then function to ameliorate diseases associated with these processes, e.g., cancer and autoimmunity.

The 2.4 Angstrom structure of β2AR bound to carazolol, described herein (PDB coordinates appear in Appendix I; SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) can be used as a model for rationally designing pharmacophore and/or candidate compounds, either de novo or by modification of known compounds. As noted below, the multiple ligand binding sites in this structure include amino acids that are highly conserved across a large number of class A G protein coupled receptors (GPCRs) indicating that the 2.4 Angstrom structure of β2AR can be used for the rational designing of ligands (e.g., therapeutic compounds) that bind to this receptor and others. Pharmacophore and candidate compounds identified through the use of the crystal structure co-ordinates will have utility as pharmaceuticals due to their ability to alter the structure and/or binding properties of β2AR. Pharmacophores and candidate compounds can be determined according to any method known in the art, including the methods described in U.S. Pat. No. 5,888,738 to Hendry, and the methods described in U.S. Pat. No. 5,856,116 to Wilson et al. the disclosures of which both are incorporated by reference in their entirety for all purposes.

The structure data provided herein can be used in conjunction with computer-modeling techniques to develop models of sites on the human β2AR or related GPCRs selected by analysis of the crystal structure data. The site models characterize the three-dimensional topography of site surface, as well as factors including van der Waals contacts, electrostatic interactions, and hydrogen-bonding opportunities. Computer simulation techniques can be used to map interaction positions for functional groups including protons, hydroxyl groups, amine groups, divalent cations, aromatic and aliphatic functional groups, amide groups, alcohol groups, etc. that are designed to interact with the model site. These groups can be designed into a pharmacophore or candidate compound with the expectation that the candidate compound will specifically bind to the site. Pharmacophore design thus involves a consideration of the ability of the candidate compounds falling within the pharmacophore to interact with a site through any or all of the available types of chemical interactions, including hydrogen bonding, van der Waals, electrostatic, and covalent interactions, although, in general, and preferably, pharmacophores interact with a site through non-covalent mechanisms.

The ability of a pharmacophore or candidate compound to bind to the human β2AR can be analyzed prior to actual synthesis using computer modeling techniques. Only those candidates that are indicated by computer modeling to bind the target with sufficient binding energy (i.e., binding energy corresponding to a dissociation constant with the target on the order of 10−2 M or tighter) can be synthesized and tested for their ability to bind to the human β2AR using binding assays or functional assays known to those of skill in the art. The computational evaluation step thus avoids the unnecessary synthesis of compounds that are unlikely to bind β2AR or one or more of its constitutive binding sites, or the related binding sites of another GPCR with adequate affinity.

A human β2AR or candidate compound(s) can be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with individual binding target sites on β2AR or binding site thereof, including, but not limited to a binding pocket of the human β2AR. One skilled in the art can use one of several methods to screen chemical entities or fragments for their ability to associate with one or more of these human β2AR binding sites. For example, increased affinity and specificity may be designed into caffeine and other xanthine molecules by combining interactions with both xanthine and non-xanthine binding sites.

The process can begin by visual inspection of, for example a target site on a computer screen, based on the human β2AR co-ordinates, or a subset of those co-ordinates (e.g., binding pocket residues V117, T118, F193, Y199, A200, W286, F289, F290, and Y316), as set forth in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). Selected fragments or chemical entities can then be positioned in a variety of orientations or “docked” within a target site of the human β2AR as defined from analysis of the crystal structure data. Docking can be accomplished using software such as Quanta (Molecular Simulations, Inc., San Diego, Calif.) and Sybyl (Tripos, Inc. St. Louis, Mo.) followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields such as CHARMM (Molecular Simulations, Inc., San Diego, Calif.), ICM (Molsoft, San Diego, Calif.), and AMBER (University of California, San Francisco).

Specialized computer programs can also assist in the process of selecting fragments or chemical entities. These include but are not limited to: GRID (Goodford, P. J., “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules,” J. Med. Chem., 28, pp. 849 857 (1985)); GRID is available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method,” Proteins: Structure, Function and Genetics, 11, pp. 29 34 (1991)); MCSS is available from Molecular Simulations, Inc., San Diego, Calif.; AUTODOCK (Goodsell, D. S, and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing,” Proteins: Structure, Function, and Genetics, 8, pp. 195 202 (1990)); AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; DOCK (Kuntz, I. D., et al. “A Geometric Approach to Macromolecule-Ligand Interactions,” J. Mol. Biol., 161, pp. 269 288 (1982)); DOCK is available from University of California, San Francisco, Calif.; CERIUS II (available from Molecular Simulations, Inc., San Diego, Calif.); and Flexx (Raret, et al. J. Mol. Biol. 261, pp. 470 489 (1996)).

After selecting suitable chemical entities or fragments, they can be assembled into a single compound. Assembly can proceed by visual inspection of the relationship of the fragments to each other on a three-dimensional image of the fragments in relation to the human β2AR or its binding sites or those of a related GPCR receptor structure or portion thereof displayed on a computer screen. Visual inspection can be followed by manual model building using software such as the Quanta or Sybyl programs described above.

Software programs also can be used to aid one skilled in the art in connecting the individual chemical entities or fragments. These include, but are not limited to CAVEAT (Bartlett, P. A., et al. “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules” In “Molecular Recognition in Chemical and Biological Problems,” Special Publ, Royal Chem. Soc., 78, pp. 182 196 (1989)); CAVEAT is available from the University of California, Berkeley, Calif.; 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.); this area is reviewed in Martin, Y. C., “3D Database Searching in Drug Design,” J. Med. Chem., 35:2145 2154 (1992)); and HOOK (available from Molecular Simulations Inc., San Diego, Calif.).

As an alternative to building candidate pharmacophores or candidate compounds up from individual fragments or chemical entities, they can be designed de novo using the structure of the β2AR, its constituent ligand binding pocket, or the homologous cavities in a related GPCR, optionally, including information from co-factor(s) or known activators or inhibitor(s) that bind to the target site. De novo design can be implemented by programs including, but not limited to LUDI (Bohm, H. J., “The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors,” J. Comp. Aid. Molec. Design, 6, pp. 61 78 (1992)); LUDI is available from Molecular Simulations, Inc., San Diego, Calif.; LEGEND (Nishibata, Y., and Itai, A., Tetrahedron 47, p. 8985 (1991); LEGEND is available from Molecular Simulations, San Diego, Calif.; and LeapFrog (available from Tripos Associates, St. Louis, Mo.).

The functional effects of known β2AR also can be altered through the use of the molecular modeling and design techniques described herein. This can be carried out by docking the structure of the known ligand on a human A2A adenosine receptor or a model structure of one or more binding sites of the human β2AR (e.g., the binding pocket described herein) and modifying the shape and charge distribution of the ligand or protein model structure to optimize the binding interactions between the ligand and protein. The modified structure can be synthesized or obtained from a library of compounds and tested for its binding affinity and/or effect on ribosome function. Of course, where the crystal structure of a complex between a human β2AR (or subunit thereof) and a ligand is known, comparisons between said complex and the structures of the present invention can be made to gain additional information about alterations in human β2AR conformation that occur upon ligand binding. This information can be used in design of optimized ligands. Compounds that interfere or activate human β2AR function (e.g., by interacting with a binding pocket) are especially well suited for the docking, co-crystallization, and optimization applications of the present invention.

Additional molecular modeling techniques also can be employed in accordance with the invention. See, e.g., Cohen, N. C., et al. “Molecular Modeling Software and Methods for Medicinal Chemistry,” J. Med. Chem., 33, pp. 883 894 (1990); Hubbard, Roderick E., “Can drugs be designed?” Curr. Opin. Biotechnol. 8, pp. 696 700 (1997); and Afshar, et al. “Structure-Based and Combinatorial Search for New RNA-Binding Drugs,” Curr. Opin. Biotechnol. 10, pp. 59 63 (1999).

Following pharmacophore or candidate compound design or selection according to any of the above methods or other methods known to one skilled in the art, the efficiency with which a candidate compound falling within the pharmacophore definition binds to the human β2AR or its ligand binding site, or alternatively binds to a related GPCR or homologous portions thereof, can be tested and optimized using computational evaluation. A candidate compound can be optimized, e.g., so that in its bound state it would preferably lack repulsive electrostatic interaction with the target site. These repulsive electrostatic interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions. It is preferred that the sum of all electrostatic interactions between the candidate compound and the human β2AR, including its ligand binding site when the candidate compound is bound to the target make a neutral or favorable contribution to the binding enthalpy or free energy.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include, but are not limited to Gaussian 92, revision C (Frisch, M. J., Gaussian, Inc., Pittsburgh, Pa. (1992)); AMBER, version 4.0 (Kollman, P. A., University of California at San Francisco, (1994)); QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. (1994)); and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif. (1994)). These programs can be run, using, e.g., a Silicon Graphics workstation, Indigo, 02-R10000 or IBM RISC/6000 workstation model 550. Other hardware and software combinations can be used to carry out the above described functions, and are known to those of skill in the art. In general, the methods described herein, particularly computer-implemented methods, comprise a step of recording or storing data onto a medium, wherein the medium can include a computer-readable medium. Additionally, or alternatively, the methods comprise a step of reporting or communicating the data to a user of interest, e.g., an operator of the device and/or computer that is employed in the method; or the computer can perform an additional useful task, e.g., alert the operator of the computer that a function has been completed, upon completing one or more determining steps of the method.

Once a pharmacophore or candidate compound has been optimally selected or designed, as described above, substitutions can then be made in some of its atoms or side groups to improve or modify its binding properties. Generally, initial substitutions are conservative in that the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Components known in the art to alter conformation should be avoided in making substitutions. Substituted candidates can be analyzed for efficiency of fit to the human β2AR (or one or more binding sites of the human β2AR) using the same methods described above.

Assays

Any one of a number of assays of function known to those of skill in the art can be used to determine the biological activity of candidate compounds.

Candidate compound interaction with the human β2AR (or one or more binding sites of human β2AR) or to a related GPCR or portion thereof can be evaluated using direct binding assays including filter binding assays, such as are known to those skilled in the art. Binding assays can be modified to evaluate candidate compounds that competitively inhibit the binding of, e.g., known human β2AR binding compounds including xanthine and xanthine-based compounds such as theophylline, theobromine and caffeine. These and other assays are described in International Publication WO 00/69391, the entire disclosure of which is incorporated by reference in its entirety for all purposes. Methods of assaying for modulators of ligand binding and signal transduction include in vitro ligand binding assays using GPCRs, such as human β2AR (or one or more binding sites selected from the binding pockets I, II and III of the human β2AR), portions thereof such as the extracellular domain, or chimeric proteins comprising one or more domains of a GPCR, oocyte GPCR expression or tissue culture cell GPCR expression, either naturally occurring or recombinant; membrane expression of a GPCR, either naturally occurring or recombinant; tissue expression of a GPCR; expression of a GPCR in a transgenic animal, etc.

As noted above, GPCRs and their alleles and polymorphic variants are G-protein coupled receptors that participate in signal transduction and are associated with cellular function in a variety of cells, e.g., neurons, immune system cells, kidney, liver, colon, adipose, and other cells. The activity of GPCR polypeptides can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding, (e.g., radioactive ligand binding), second messengers (e.g., cAMP, cGMP, IP3, DAG, or Ca2+), ion flux, phosphorylation levels, transcription levels, neurotransmitter levels, and the like. Such assays can be used to test for inhibitors and activators of a GPCR. In particular, the assays can be used to test for compounds that modulate natural ligand-induced GPCR activity, for example, by modulating the binding of the natural ligand to the receptor and/or by modulating the ability of the natural ligand to activate the receptor. Typically in such assays, the test compound is contacted with the GPCR in the presence of the natural ligand. The natural ligand can be added to the assay before, after, or concurrently with the test compound. The results of the assay, for example, the level of binding, calcium mobilization, etc. is then compared to the level in a control assay that comprises the GPCR and natural ligand in the absence of the test compound.

Screening assays of the invention are used to identify modulators that can be used as therapeutic agents, e.g., antagonists of GPCR activity. For example, carazolol is a known high-affinity inverse agonist of human β2AR.

The effects of test compounds upon the function of the GPCR polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the GPCRs and natural ligand-mediated GPCR activity. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca2+, IP3 or cAMP.

For a general review of GPCR signal transduction and methods of assaying signal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature 10:349:117-27 (1991); Bourne et al., Nature 348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem. 67:653-92 (1998).

Modulators of GPCR activity are tested using GPCR polypeptides, either recombinant or naturally occurring. The protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring. For example, neurons, cells of the immune system, adipocytes, kidney cells, transformed cells, or membranes can be used. Modulation is tested using one of the in vitro or in vivo assays described herein or others as generally known in the art. Signal transduction can also be examined in vitro with soluble or solid state reactions, using a chimeric molecule such as an extracellular domain of a receptor covalently linked to a heterologous signal transduction domain, or a heterologous extracellular domain covalently linked to the transmembrane and or cytoplasmic domain of a receptor. Furthermore, ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding.

Ligand binding to a human β2AR (or one or more binding sites thereof) or a chimeric protein derivative can be tested in a number of formats. For example, binding can be performed in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Typically, in an assay of the invention, the binding of the natural ligand to its receptor is measured in the presence of a candidate modulator. Alternatively, the binding of the candidate modulator can be measured in the presence of the natural ligand. Often, competitive assay that measure the ability of a compound to compete with binding of the natural ligand to the receptor are used. Binding can be measured by assessing GPCR activity or by other assays: binding can be tested by measuring e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape) changes, or changes in chromatographic or solubility properties.

Receptor-G-protein interactions can also be used to assay for modulators. For example, in the absence of GTP, binding of an activator such as the natural ligand will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways, as noted above. Such an assay can be modified to search for inhibitors. For example, a ligand can be added to the human β2AR and G protein in the absence of GTP to form a tight complex. Inhibitors can be identified by looking at dissociation of the receptor-G protein complex. In the presence of GTP, release of the alpha subunit of the G protein from the other two G protein subunits serves as a criterion of activation.

An activated or inhibited G-protein will in turn alter the properties of downstream effectors such as proteins, enzymes, and channels. The classic examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G-protein, phospholipase C by Gq and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins. Downstream consequences such as generation of diacyl glycerol and IP3 by phospholipase C, and in turn, for calcium mobilization e.g., by IP3 can also be examined. Thus, modulators can be evaluated for the ability to stimulate or inhibit ligand-mediated downstream effects. In other examples, the ability of a modulator to activate a GPCR expressed in adipocytes in comparison to the ability of a natural ligand, can be determined using assays such as lipolysis (see, e.g., WO01/61359).

Activated GPCRs become substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well). Thus, activators will promote the transfer of 32P from gamma-labeled GTP to the receptor, which can be assayed with a scintillation counter. The phosphorylation of the C-terminal tail will promote the binding of arrestin-like proteins and will interfere with the binding of G-proteins. The kinase/arrestin pathway plays a key role in the desensitization of many GPCR receptors. Modulators can therefore also be identified using assays involving beta-arrestin recruitment. Beta-arrestin serves as a regulatory protein that is distributed throughout the cytoplasm in unactivated cells. Ligand binding to an appropriate GPCR is associated with redistribution of beta-arrestin from the cytoplasm to the cell surface, where it associates with the GPCR. Thus, receptor activation and the effect of candidate modulators on ligand-induced receptor activation, can be assessed by monitoring beta-arrestin recruitment to the cell surface. This is frequently performed by transfecting a labeled beta-arrestin fusion protein (e.g., beta-arrestin-green fluorescent protein (GFP)) into cells and monitoring its distribution using confocal microscopy (see, e.g., Groarke et al., J. Biol. Chem. 274(33):23263-69 (1999)).

Receptor internalization assays can also be used to assess receptor function. Upon ligand binding, the G-protein coupled receptor—ligand complex is internalized from the plasma membrane by a clathrin-coated vesicular endocytic process; internalization motifs on the receptors bind to adaptor protein complexes and mediate the recruitment of the activated receptors into clathrin-coated pits and vesicles. Because only activated receptors are internalized, it is possible to detect ligand-receptor binding by determining the amount of internalized receptor. In one assay format, cells are transiently transfected with radiolabeled receptor and incubated for an appropriate period of time to allow for ligand binding and receptor internalization. Thereafter, surface-bound radioactivity is removed by washing with an acid solution, the cells are solubilized, and the amount of internalized radioactivity is calculated as a percentage of ligand binding. See, e.g., Vrecl et al., Mol. Endocrinol. 12:1818-29 (1988) and Conway et al., J. Cell Physiol. 189(3):341-55 (2001). In addition, receptor internalization approaches have allowed real-time optical measurements of GPCR interactions with other cellular components in living cells (see, e.g., Barak et al., Mol. Pharmacol. 51(2)177-84 (1997)). Modulators can be identified by comparing receptor internalization levels in control cells and cells contacted with candidate compounds. For example, candidate modulators the human β2AR are assayed by examining their effects on receptor internalization upon binding of the natural ligand.

Another technology that can be used to evaluate GPCR-protein interactions in living cells involves bioluminescence resonance energy transfer (BRET). A detailed discussion regarding BRET can be found in Kroeger et al., J. Biol. Chem., 276(16):12736-43 (2001).

Receptor-stimulated guanosine 5′-O-(γ-Thio)-Triphosphate ([35S]GTPγS) binding to G-proteins can also be used as an assay for evaluating modulators of GPCRs. [35S]GTPγS is a radiolabeled GTP analog that has a high affinity for all types of G-proteins, is available with a high specific activity and, although unstable in the unbound form, is not hydrolyzed when bound to the G-protein. Thus, it is possible to quantitatively assess ligand-bound receptor by comparing stimulated versus unstimulated [35S]GTPγS binding utilizing, for example, a liquid scintillation counter. Inhibitors of the receptor-ligand interactions would result in decreased [35S]GTPγS binding. Descriptions of [35S]GTPγS binding assays are provided in Traynor and Nahorski, Mol. Pharmacol. 47(4):848-54 (1995) and Bohn et al., Nature 408:720-23 (2000).

The ability of modulators to affect ligand-induced ion flux can also be determined. Ion flux can be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing a GPCR. One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents are conveniently determined using the standard methodology (see, e.g., Hamil et al., Pflügers. Archiv. 391:85 (1981). Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Gonzales & Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70 (1994)). Generally, the compounds to be tested are present in the range from 1 pM to 100 mM.

Preferred assays for G-protein coupled receptors include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G-protein coupled receptors and the natural ligands disclosed herein as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage are monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that can be employed are those disclosed in the Molecular Probes 1997 Catalog. For G-protein coupled receptors, promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049-10053 (1991)). Such promiscuous G-proteins allow coupling of a wide range of receptors to signal transduction pathways in heterologous cells.

Receptor activation by ligand binding typically initiates subsequent intracellular events, e.g., increases in second messengers such as IP3, which releases intracellular stores of calcium ions. Activation of some G-protein coupled receptors stimulates the formation of inositol triphosphate (IP3) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)). IP3 in turn stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G-protein coupled receptor function. Cells expressing such G-protein coupled receptors can exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it can be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.

Other assays can involve determining the activity of receptors which, when activated by ligand binding, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting downstream effectors such as adenylate cyclase. There are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci. U.S.A. 88:9868-9872 (1991) and Dhallan et al., Nature 347:184-187 (1990)). In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it can be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay. Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-gated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.

In one embodiment, changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offermanns & Simon, J. Biol. Chem. 270:15175-15180 (1995) can be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994) can be used to determine the level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Pat. No. 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with 3H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates are separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist to cpm in the presence of buffer control (which can or can not contain an agonist).

In another embodiment, transcription levels can be measured to assess the effects of a test compound on ligand-induced signal transduction. A host cell containing the protein of interest is contacted with a test compound in the presence of the natural ligand for a sufficient time to effect any interactions, and then the level of gene expression is measured. The amount of time to effect such interactions can be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription can be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest can be detected using northern blots or their polypeptide products can be identified using immunoassays. Alternatively, transcription based assays using reporter genes can be used as described in U.S. Pat. No. 5,436,128, herein incorporated by reference. The reporter genes can be, e.g., chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, beta-galactosidase and alkaline phosphatase. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it can be compared with the amount of transcription in a substantially identical cell that lacks the protein of interest. A substantially identical cell can be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the protein of interest.

Samples that are treated-with a potential GPCR inhibitor or activator are compared to control samples comprising the natural ligand without the test compound to examine the extent of modulation. Control samples (untreated with activators or inhibitors) are assigned a relative GPCR activity value of 100. Inhibition of a GPCR is achieved when the GPCR activity value relative to the control is about 90%, optionally 50%, optionally 25-0%. Activation of a GPCR is achieved when the GPCR activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.

In one embodiment the invention provides soluble assays using molecules such as a domain, e.g., a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; a GPCR; or a cell or tissue expressing a GPCR, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, GPCR, or cell or tissue expressing a GPCR is attached to a solid phase substrate.

Certain screening methods involve screening for a compound that modulates the expression of the GPCRs described herein, or the levels of natural ligands, e.g., ASP and stanniocalcins. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing the GPCR or ligand and then detecting an increase or decrease in expression (either transcript or translation product). Such assays are typically performed with cells that express the endogenous GPCR or ligand.

Expression can be detected in a number of different ways. As described herein, the expression levels of the protein in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of the GPCR or protein ligand. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques (see above). Alternatively, protein can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to the protein.

Other cell-based assays are reporter assays conducted with cells that do not express the protein. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a promoter that is operably linked to a reporter gene that encodes a detectable product. A number of different reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes. Exemplary enzyme reporters include, but are not limited to, beta-glucuronidase, CAT (chloramphenicol acetyl transferase), luciferase, beta-galactosidase and alkaline phosphatase.

In these assays, cells harboring the reporter construct are contacted with a test compound. A test compound that either modulates the activity of the promoter by binding to it or triggers a cascade that produces a molecule that modulates the promoter causes expression of the detectable reporter. Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of the GPCR or ligand and a reporter operably linked thereto. Here, too, an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression, can be identified by the generation of signal associated with reporter expression.

In one embodiment the invention provides soluble assays using molecules such as a domain, e.g., a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; a GPCR; or a cell or tissue expressing a GPCR, either naturally occurring or recombinant.

In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, GPCR, or cell or tissue expressing a GPCR is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100-1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention.

The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the signal transduction molecule of interest) is attached to the solid support by interaction of the tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and are appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.

Modulators

Inhibitors and/or activators identified according to the methods of the invention can be provided from libraries of compounds available from a number of sources or can be derived by combinatorial chemistry approaches known in the art. Such libraries include but are not limited to the available Chemical Director, Maybridge, and natural product collections. In one embodiment of the invention libraries of compounds with known or predicted structures can be docked to the human β2AR structures of the invention. In another embodiment, the libraries for ligands binding to the ligand binding site can include carazolol and related compounds. In another embodiment, the libraries can include a linker component or moiety. In some embodiments, the linker can include from about 10-22 atoms and can include one or more of C, O, N, S, and/or H atoms. In another embodiment, the libraries can include a ligand binding site (also known as the ligand, agonist, or antagonist binding pocket) component or moiety. In some embodiments, the libraries can include drug-like molecules, i.e., molecules having structural attributes of one or more compounds known to bind to and/or affect a physiologic function of a GPCR.

In some embodiments, the invention includes compounds that can be tested as modulators of GPCR activity. Compounds tested as modulators of GPCRs can be any small chemical compound or biological entity. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps. The assays are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such “combinatorial chemical libraries” or ligand libraries are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Russell & Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

It is noted that modulators that compete with the binding and/or activity of the known ligands for to human β2AR can be used to treat various diseases including, but not limited to, coronary artery disease, atherosclerosis, thrombosis, obesity, diabetes, stroke, and other diseases.

In one embodiment, a modulator binds to a site on a GPCR, e.g., a human β2AR. In one aspect, the site is a carazolol binding site. In a related aspect, the site is a ligand binding site. In another aspect, the modulator has a first moiety that binds to a binding site. In another aspect, the first moiety is connected to a linker. In another aspect, the first moiety and the linker are connected to at least one additional moiety that binds to a site other than that bound by the first moiety. In another aspect, the two or more moieties are not connected by a linker and are both present in a composition.

Computer-Based Modeling of β2AR

Protein-ligand docking aims to employ principles by which protein receptors, e.g., human β2AR, recognize, interact, and associate with molecular substrates and compounds to predict the structure arising from the association between a given compound and a target protein of known three-dimensional structure.

In protein-ligand docking, the search algorithm can allow the degrees of freedom of the protein-ligand system to be sampled sufficiently as to include the true binding modes. Three general categories of algorithms have been developed to address this problem of ligand flexibility: systematic methods; random or stochastic methods; and simulation methods.

Systematic search algorithms attempt to explore all degrees of freedom in a molecule. These algorithms can be further divided into three types: conformational search methods, fragmentation methods, and database methods.

In conformational search methods, all rotatable bonds in the ligand are systematically rotated through 360° using a fixed increment, until all possible combinations have been generated and evaluated. As the number of structures generated increases immensely with the number of rotatable bonds (combinatorial explosion), the application of this type of method, in its purest form, is very limited.

Fragmentation methods use two different approaches to incrementally grow the ligands into the active site. One approach is by docking the several fragments into a site and linking them covalently to recreate the initial ligand (“the place-and-join approach”). Another approach is by dividing the ligand into a rigid core-fragment that is docked in first place and flexible regions that are subsequently and successively added (“the incremental approach”). DOCK (see above) is an example of s docking programs that use a fragmentation search method.

Database methods using libraries of pre-generated conformations or conformational ensembles to address the combinatorial explosion problem. A example of a docking program using database methods is FLOG which generates a small set of 25 database conformations per molecule based on distance geometry, that are subsequently subject to a rigid docking protocol.

Random search algorithms sample the conformational space by performing random changes to a single ligand or a population of ligands. At each step, the alteration performed is accepted or rejected based on a predefined probability function. There are three basic types of methods based on random algorithms: Monte Carlo methods (MC), Genetic Algorithm methods (GA), and Tabu Search methods.

Simulation methods employ a rather different approach to the docking problem, based on the calculation of the solutions to Newton's equations of motion. Two major types exist: molecular dynamics (MD) and pure energy minimization methods.

Scoring functions normally employed in protein-ligand docking are generally able to predict binding free energies within 7-10 kJ/mol and can be divided into three major classes: force field-based, empirical, and knowledge-based scoring functions.

In force-field based scoring, standard force fields quantify the sum of two energies: the interaction energy between the receptor and the ligand, and the internal energy of the ligand. The energies are normally accounted through a combination of a van der Waals with an electrostatic energy terms. A Lennard-Jones potential is used to describe the van der Waals energy term, whereas the electrostatic term is given by a Coulombic formulation with a distance-dependent dielectric function that lessens the contribution from charge-charge interactions.

Empirical scoring functions are based on the idea that binding energies can be approximated by a sum of several individual uncorrelated terms. Experimentally determined binding energies and sometimes a training set of experimentally resolved receptor-ligand complexes are used to determine the coefficients for the various terms by means of a regression analysis.

Knowledge-based scoring functions focus on following the rules and general principles statistically derived that aim to reproduce experimentally determined structures, instead of binding energies, trying to implicitly capture binding effects that are difficult to model explicitly. Typically, these methods use very simple atomic interactions-pair potentials, allowing large compound databases to be efficiently screened. These potentials are based on the frequency of occurrence of different atom-atom pair contacts and other typical interactions in large datasets of protein-ligand complexes of known structure. Therefore, their derivation is dependent on the information available in limited sets of structures.

Consensus Scoring combines the information obtained from different scores to compensate for errors from individual scoring functions, therefore improving the probability of finding the correct solution. Several studies have demonstrated the success of consensus scoring methods in relation to the use of individual functions schemes.

Using the Protein-ligand docking methods described above, a predicted association can be made between a selected chemical library compound (see above for examples) and the binding sites in the human β2AR structure described in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). These methods will therefore allow the generation of a binding profile for any known compound in any of the binding sites or cavities of the human β2AR based on the simulated docking of the compound.

In another embodiment, a form of computer-assisted drug design is employed in which a computer system is used to generate a three-dimensional structure of the candidate class A GPCR based on the structural information encoded by the amino acid sequence. This will allow use of the methods described above to identify candidate compounds based on their ability to dock in one or more of the predicted GPCR structure binding sites. In one aspect, the input amino acid sequence of the GPCR interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the class A GPCR. The models of the class A GPCR structure are then examined to identify the position and structure of the binding sites, e.g., a binding pocket. The position and structure of the predicted binding site(s) is then used to identify various compounds that modulate ligand-receptor binding using the methods described above.

The three-dimensional structural model of the GPCR is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a GPCR polypeptide into the computer system. The amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein. At least 10 residues of the amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM. The three-dimensional structural model of the GPCR is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art. Any method of protein structure modeling such as ab-initio modeling, threading or sequence-sequence based methods of fold recognition. In one embodiment, the AS2TS system of protein structure modeling is used. In other embodiments, a sequence alignment in combination with a threshold protein sequence similarity to determine a set of protein sequences for which to model protein structure is used. In one aspect, sequence alignments are generated for the set of sequences to be modeled with sequences of proteins with solved empirical structure in a protein structure databank known to one of skill in the art. If the sequences to be modeled have a sufficient similarity to one or more sequences with known protein structure, then the three dimensional structure of the sequence can be modeled.

The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the GPCR of interest. In one embodiment, software can look at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as “energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.

The tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure. The user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.

In another embodiment, protein structure alignments can be used to determine the structure of GPCRs using the known structure of the β2AR (Appendix I) (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). Protein structure alignments preferably are sets of correspondences between spatial co-ordinates of sets of carbon alpha atoms which form the ‘backbone’ of the three-dimensional structure of polypeptides, although alignments of other backbone or side chain atoms also can be envisioned. These correspondences are generated by computationally aligning or superimposing two sets of atoms order to minimize distance between the two sets of carbon alpha atoms. The root mean square deviation (RMSD) of all the corresponding carbon alpha atoms in the backbone is commonly used as a quantitative measure of the quality of alignment. Another quantitative measure of alignment is the number of equivalent or structurally aligned residues.

In another embodiment, a GPCR structure is calculated based on the solved structure of the human β2AR by computationally aligning or superimposing two sets of atoms to minimize distance between the two sets of carbon alpha atoms (i.e., the alpha carbon atoms of the human β2AR and an unknown GPCR structure), followed by one or more of simulated annealing and energy minimization. The result of this calculation is a computed structure for a GPCR that provides atomic co-ordinates for the alpha carbon backbone as well as side chain atoms.

A variety of methods for generating an optimal set of correspondences can be used in the present invention. Some methods use the calculation of distance matrices to generate an optimal alignment. Other methods maximize the number of equivalent residues while RMSD is kept close to a constant value.

In the calculation of correspondences, various cutoff values can be specified to increase or decrease the stringency of the alignment. These cutoffs can be specified using distance in Angstroms. Depending on the level of stringency employed in the present invention, the distance cutoff used is less than 10 Angstroms or less than 5 Angstroms, or less than 4 Angstroms, or less than 3 Angstroms. One of ordinary skill will recognize that the utility of stringency criterion depends on the resolution of the structure determination.

In another embodiment of the present invention, the set of residue-residue correspondences are created using a local-global alignment (LGA), as described in US Patent Publication Number 2004/0185486. In this method, a set of local superpositions are created in order to detect regions which are most similar. The LGA scoring function has two components, LCS (longest continuous segments) and GDT (global distance test), established for the detection of regions of local and global structure similarities between proteins. In comparing two protein structures, the LCS procedure is able to localize and superimpose the longest segments of residues that can fit under a selected RMSD cutoff. The GDT algorithm is designed to complement evaluations made with LCS searching for the largest (not necessary continuous) set of ‘equivalent’ residues that deviate by no more than a specified distance cutoff.

Using the protein structure alignments described above, the structure of human β2AR in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) can be used as a model on which to discern the structure of other GPCRs and/or their predicted ligand-binding sites.

Once the GPCR structure has been generated, a binding pocket can be identified by the computer system. Computational models seek to identify the regions by characterization of the three dimensional structure of the GPCR. Some methods of identifying a binding pocket use triangulation such as weighted Delaunay triangulation to determine pocket volumes (castP). Other methods use spheres to determining protein pocket volumes (Q-site-finder, UniquePocket). Conserved binding-site identification seeks to identify conserved regions such as a binding pocket through associating the residues which form the aforementioned regions with conserved residues in homologous protein sequences or structures, e.g., through the use of sequence alignments.

One method of identifying a binding pocket in a GPCR entails filling the three dimensional protein structures with spheres, creating a “negative image” of the structure. A cutoff distance, such as 8 Angstroms, is used to determine spheres which interact with residues. Spheres are labeled as conserved or not-conserved based on their interaction with residues which form a conserved binding site. The conserved spheres are clustered based on their three dimensional co-ordinates to identify a set of spheres with interact with conserved residues and are proximal in three dimensional space forming a cluster. Three-dimensional structures for potential compounds are generated by entering chemical formulas of compounds. The three-dimensional structure of the potential compound is then compared to that of the GPCR protein ligand-binding site(s) (e.g., a binding pocket) to identify compounds that bind to the GPCR binding site(s). Binding affinity between the GPCR binding site(s) and the compound is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.

It should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and can not have been selected to delineate or circumscribe the inventive subject matter. Accordingly, the disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the claims.

Reference will now be made in detail to particularly preferred embodiments of the invention. Examples of the preferred embodiments are illustrated in the following Examples section.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols. A and B (1992).

Example 1

Crystallization of a β2AR Protein Using a Lipidic Cubic Phase/Sterol Method

This Example describes the generation of diffraction-quality crystals of a β2AR protein, specifically crystals of β2AR-T4L, a fusion protein of human β2AR with T4 lysozyme, bound to carazolol. A detailed description of the protein and its synthesis is provided in Example 4. Briefly, T4 lysozyme was generated by three distinct modifications to β2AR: (1) a fusion protein was created by replacement of the third intracellular loop with T4L, (2) the carboxyl terminal 48 amino acids were deleted, and (3) a glycosylation site at Asn187 was eliminated through a glutamate substitution. β2AR-T4L was expressed in Sf9 insect cells, solubilized in 1% dodecylmaltoside, and purified by sequential antibody and ligand affinity chromatography.

LCP Crystallization Setup

Protein solution (30 mg/ml) was mixed with a host lipid or lipid mixture typically in 2:3 ratio by volume ratio using a syringe mixer (Cheng, et al., Chem Phys Lipids 95, 11 (1998). Upon mixing (˜100 passages, 2-3 min) the sample spontaneously formed homogeneous transparent cubic phase, which was directly used in crystallization trials. Robotic trials were performed using an in meso crystallization robot (Cherezov, et al., Acta Cryst D 60, 1795 (2004)). Six-well glass sandwich plates (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov, J Appl Cryst 36, 1372 (2003)), were filled with 25 or 50 nL protein-laden lipidic cubic phase drops overlaid by 800 μL, of precipitant solution in each well and sealed with a glass coverslip. Manual setups were performed in Impact microbatch plates (Hampton Research cat#HR3-293), Innovaplate SD-2 sitting drop plates (Hampton Research cat#HR3-083) or VDX48 hanging drop plates (Hampton Research cat#HR3-275). Modified repetitive syringe dispenser (Cherezov, et al., J Appl Cryst 38, 398 (2005)), coupled with a gas-tight 10 μL syringe was used to deliver 70 nL of cubic phase drops per well to which 1-2 μL of precipitant solution was added with a microvolume pipette. Reservoirs of the Innovaplate and VDX48 plates were filed with 50 and 100 μL of precipitant respectively. All operations starting from mixing lipid and protein were performed at room temperature (˜21-23° C.). After setup, plates were transferred into an automatic incubator/imager (Rocklmager 1000, Formulatrix Inc.) maintained at 20° C. Plates were imaged every 12 hours for the first 3 days, then every day until 7 days and after that on the 10th and on the 14th day.

Initial Hits

Initial trials were performed using protein solution at a concentration of 30 mg/mL mixed with monoolein as a host lipid against a set of 6 commercial screens (Index HT, SaltRx HT and MemFac HT from Hampton Research, JCSG+ and MbClass from Nextal, and MemSys&MemStart from Molecular Dimensions) set up in duplicates. Initial hits detected in three different wells contained extremely small, <5 μm, needle-like birefringent crystal showers. The detection of such small colorless protein crystals in LCP was made feasible by specially developed glass sandwich plates with optimized optical properties (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov et al., J Appl Cryst 36, 1372 (2003)). Hit conditions were similar by chemical composition containing 30% v/v PEG 400 as a precipitant, low concentration of Li sulfate and a buffer at pH 6.5 or 7.5.

Optimization

Crystal condition optimization is well known to practioners in the art of x-ray crystallography. What follows is a specific example of a generally applicable optimization approach in which one or more of the crystallization mixture components is systematically varied in concentration or substituted by a chemical analog. Initial rounds of optimization were focused on varying concentration of the main precipitant, PEG 400, buffer pH and identity, and salt identity and concentration. As a result, Li sulfate was replaced with Na sulfate and useful concentration and pH ranges were established. Crystals were still rather small reaching ˜15×5×1 μm in size.

Further, lipid and soluble additives were searched for and optimized simultaneously. Three different host lipids (monopalmitolein, monovaccenin and monoolein), five lipid additives to monoolein host (DOPE, DOPE-Me, DOPC, Asolectin and cholesterol) and 96-well soluble additives screen were tried in different combinations along with the previously found basic crystallization conditions. One of the soluble additives, 1,4-butanediol, stood out, but only when it was used in combination with lipid additives, DOPE or cholesterol. When DOPE was used as an additive the crystals grew as thin plates (40×7×2 μm), while when cholesterol was used the crystals grew as small rods (30×5×5 μm). DOPE was dropped out in favor of cholesterol in the subsequent optimization steps.

Final optimization required fine tuning concentrations of all components (protein, PEG 400, Na sulfate, 1,4-butanediol, cholesterol, buffer pH). At the final stages of optimization, higher concentrations of protein, e.g., 50 mg/mL protein solution, were preferred. Decreasing the volume of lipidic cubic phase per trial from 50 to 20 nL consistently produced larger crystals. The best crystals (40×20×5 μm; FIG. 1) were obtained in 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid. Thus, in another aspect, the invention provides a method of directly adding a lipid additive (e.g., cholesterol, DOPE) to the host lipid prior to combining with the protein mixture, thereby significantly improving the size and quality of LCP grown crystals. Previously, exogenous lipid had been added only to the protein solution prior to combining the protein solution with the host lipid (Luecke, et al., Science 293, 1499 (2001)).

To limit the range of precipitant and additives concentrations used for screening, previously published phased diagrams were used, e.g., for monoolein (Qiu, et al., Biomaterials 21, 223 (2000)), and monovaccenin (Qiu, et al., J. Phys. Chem. B 102, 4819 (1998)), and the effects on monoolein phase behavior of soluble (Cherezov, et al., Biophys J 81, 225 (2001)), and lipid additives (Cherezov, et al., Biophys J 83, 3393 (2002)). Certain lipid mixtures required pre-screening their phase behavior by microscope visualization and by X-ray, prior to being mixed with the protein for crystallization trials. Thirty-three 96-well optimization screens were used in combinations with different lipid mixtures, resulting in over 15,000 trials. This throughput was made feasible through the use of an in meso crystallization robot and automatic imager.

Typically, the best crystals grown under lipidic cubic phase conditions appear at the boundary between the cubic and the sponge phase (Cherezov, et al., J Mol Biol 357, 1605 (2006); Wadsten, et al., J Mol Biol 364, 44 (2006)). When crystals are close to the phase boundary, equally good crystals are obtained in either phase.

Crystal Harvesting

Crystals were harvested directly from the glass sandwich plates (FIG. 2) because this method provided results superior to those obtained with microbatch or vapor diffusion plates. These plates have been specifically designed to perform optimally only at the screening and optimization stages (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov and M. Caffrey, J Appl Cryst 36, 1372 (2003)). No harvesting has been previously attempted from them, due to the difficulties in separating glass slides strongly bound by a high performance double sticky tape. Thus, in another aspect, the invention disclosed herein provides a special technique for opening individual wells and harvesting crystals from them. A corner of a capillary cutting stone (in this instance, from Hampton Research) was used to scratch the top glass around the perimeter of the well. Gently pressing the glass slide just outside the scratch allows propagation of the scratch through the depth of the glass. The glass slide was then broken in one point just outside of the scratched perimeter using a sharp needle. This hole was used to lift up the glass slide and expose the cubic phase for harvesting. An extra drop of ˜5 μL of precipitant was added to the well to reduce dehydration. Using this technique, it was possible to open up and harvest crystals successfully from more than 80% of attempted wells.

Crystals were scooped directly from the lipidic cubic phase using 30 μm aperture MiTeGen MicroMounts and plunged into liquid nitrogen. Care was taken to drag as little as possible lipid around the crystal to decrease the unwanted background. Attempts to dissolve the lipids, either by increasing concentration of PEG400 or using a mineral oil, typically resulted in decreasing the diffraction power of the crystals.

Data Collection

During screening for diffraction of these crystals at APS beamline GM/CA CAT (FIG. 3), the crystals themselves could not be observed in the mounted loops (FIG. 1a). Therefore, a systematic screening of the loop material with varying beams was conducted to identify the crystal in the loop. Optimization of the diffraction with a low x-ray dose was then used to center the crystals and eventually allow for complete data collection using the 10 μm×10 μm minibeam setup at GM/CA CAT. The complete data set is then compared to data filtered by a sigma cutoff (see Table 1). All of the data was used in structure solution and refinement.

TABLE 1
# Observed# Unique
ResolutionreflreflRedundancyCompletenessRsymI/SIGMAR-measRmrgd-F
Signal/noise −3
10  335233310.187.40%6.60%23.256.90%2.70%
8  359135410.199.40%7.60%22.68.00%2.80%
6  10480100310.499.60%9.90%20.0110.50%4.00%
3  1260081196810.599.80%13.30%14.0114.00%6.60%
2.833158313010.6100.10%38.00%6.4239.90%18.70%
2.719702189310.499.70%49.50%4.9652.10%24.90%
2.623772227510.499.90%60.20%4.0763.40%30.10%
2.51410825585.599.30%58.90%2.6965.10%51.80%
2.41467230604.899.10%67.80%2.1875.70%62.80%
total248843265749.499.50%12.70%9.6213.40%11.40%
Signal/noise 0
10  335233310.187.40%6.60%23.256.90%2.70%
8  359135410.199.40%7.60%22.68.00%2.80%
6  10480100310.499.60%9.90%20.0110.50%4.00%
3  1256281192310.599.40%13.30%14.0614.00%6.50%
2.832679307710.698.40%37.60%6.5439.50%17.70%
2.719346184910.597.40%48.80%5.0851.30%23.40%
2.623201221010.597.00%58.90%4.262.00%28.00%
2.51346124065.693.40%56.40%2.8862.30%45.90%
2.41383328274.991.50%64.10%2.471.50%54.30%
total245571259829.597.30%12.60%9.8513.30%10.80%

Example 2

Using the LCP/Sterol Method to Generate Additional Membrane Protein Crystals

In addition to the β2AR-T4L/carazolol structure (Examples 1, 3, and 4), the LCP/sterol matrix has successfully been used to crystallize a diversity of receptor-ligand systems.

1. β2AR-T4L(E122W)

A thermally-stabilized construct of β2AR-T4L comprising an E122W mutation has been crystallized in the presence of both agonist and antagonist ligands including: alprenolol, timolol, clenbuterol and carazolol. For lipidic cubic phase (LCP) crystallization of NAR-T4L(E122W), robotic trials were performed using an in meso crystallization robot (Cherezov et al., 2004). Glass sandwich plates in 96-well format (Cherezov and Caffrey, 2003; Cherezov et al., 2004) were filled with 25 or 50 nL protein-laden LCP drops overlaid by 0.8 μL of precipitant solution in each well and sealed with a glass coverslip. All operations starting from mixing lipid and protein were performed at room temperature (˜21-23° C.). Crystals were obtained in 28% (v/v) PEG 400, 0.3 M potassium formate, 0.1 M Bis-tris propane pH 7.0 and saturating concentrations of ligand (e.g., 2 mM in the case of timolol) using 10% (w/w) cholesterol in monoolein as the host lipid. Diffraction data were collected on all four ligand complexes (see FIG. 4), and structures were determined for alprenolol (3.2 Å), timolol (2.8 Å), and carazolol (2.8 Å).

2. A2AR-T4L

The applicability of the monoolein cholesterol system in the crystallization of non-biogenic amine receptors has also been demonstrated with the structural determination of the human A2A adenosine receptor (A2AR-T4L) bound to a high affinity selective antagonist, ZM241385, to 2.6 Å resolution. See FIG. 4. For lipidic cubic phase (LCP) crystallization of the human A2A adenosine receptor in meso, glass sandwich plates (Cherezov, et al., Acta Crystallogr D Biol Crystallogr, 60, 1795 (2004)) were filled with 50 nl receptor-cholesterol-monoolein LCP drops overlaid by 0.8 μl of precipitant solution in each well and sealed with a glass coverslip. Lipid:receptor LCP mixture typically contained monoolein:cholesterol (54%:6% (w/w)) and receptor (40% (w/w)). Crystallization set-ups were performed at ambient temperature (22±2° C.). Plates were incubated and imaged at 20° C. using an automated incubator/imager (RockImager 1000, Formulatrix). Data-collection quality crystals 100 μm×10 μm×5 μm) were obtained in 30% (v/v) PEG 400 (range of 28-32%), 186 mM Lithium sulfate (range of 180 to 220 mM), 100 mM Sodium citrate (pH 6.5) (Range of 5.5 to 6.5) and 200 μM ZM241385. The protein crystallized in the primitive monoclinic space group P21 with one molecule per asymmetric unit and an estimated solvent content of 52%.

3. β2AR(E122W)

Initial crystals of β2AR(E122W), lacking inserted T4 lysozyme, have also been obtained. The protein was extracted from insect cell membranes using a mixture of 0.5% w/v dodecyl maltoside (DDM), 0.1% w/v cholesteryl hemisuccinate (CHS) and 1 mM timolol. Timolol was maintained at 1 mM throughout the first steps of the purification. The extracted protein was purified by binding overnight to Talon™ immobilized metal affinity resin followed by a standard washing and elution with 200 mM imidazole. Adenosine triphosphate at 5 mM in combination with 10 mM MgCl2 was used to eliminate chaperone protein contamination. Eluted protein was concentrated to 2.5 mL and desalted into a 0 mM imidazole buffer using a PD10 desalting column (GE-Biosciences). Protein was then bound to 100 μL of Ni-sepharose immobilized metal affinity resin in the presence of PNGase (New England Biolabs) to remove glycosylation, and incubated overnight. After incubation the protein was washed on the column and timolol was replaced by carazolol for structure solution. The protein bound to carazolol was eluted from the Ni-Sepharose column, treated with 100 mM Nacitrate pH 7.5, and concentrated to 50 mg/mL.

The protein solution was then mixed with monoolein containing 10% cholesterol at a ration of 40:60% w/w protein to lipid to generate the lipidic cubic phase used in crystallization trials. The LCP lipid containing protein was dispensed onto glass sandwich crystallization plates at a volume of 20 nL to which 1 μL of crystallization solution was added. The entire experiment in 96 well format was then covered by an additional glass plate which was fastened to the first by virtue of double back sticky tape. Initial crystals have been obtained after 24 hours by addition of a solution containing 35% v/v PEG 400, 100 mM NaSO4 100 mM Bis tris propane pH 7 and 8% 2,6 hexanediol.

By way of a prophetic example, optimized crystals of β2AR(E122W) obtained by this method are screened for their ability to diffract at high resolution, e.g., less than 3.5 Å or, more preferably, less than 3 Å. Guidance for optimization is provided by the optimization protocols set forth herein and in the examples. In combination with the teaching provided herein, one skilled in the art will readily identify appropriate beam settings to obtain diffraction patterns from which a detailed molecular structure of the optimally crystallized protein can be solved.

4. CXCR4-T4L

CXCR4, also called fusin, is a GPCR protein specific for stromal-derived-factor-1 (SDF-1 also called CXCL12), a molecule endowed with potent chemotactic activity for lymphocytes. This Example teaches prophetically how the methods of the invention may be used to generate diffraction quality crystals of a fusion protein comprising CXCR4 (CXCR4-T4L).

The cDNA encoding CXCR4 is synthesized by outsourcing to DNA2.0 where the DNA was optimized for human codon usage, elimination of transcribed RNA secondary structure, elimination of ribosome binding sites and avoidance of common restrictions sites used in subsequent cloning procedures. Two initial variants are contracted to be synthesized, the first encoding a wild-type full-length receptor and the second a full length receptor with a fusion protein located between transmembrane helix V and helix VI, effectively eliminating the third intracellular loop (3IL) region of the receptor. In the case of CXCR4, T4-lysozyme (T4L) is the fusion protein fused in the place of the 3IL. A set of guidelines is followed for the incorporation of T4L into the fusion protein which minimizes the possibility of structural disruption and concomitant effects on protein expression and stability. Briefly, the 5′ insertion point for the fusion protein takes precedence over the 3′ insertion point and is located 66 nucleotides (22 residues) downstream of the codon for a conserved proline on helix V of the receptor. If the 3IL section of the receptor is large, the 3′ fusion point is set 87 nucleotides (29 residues) upstream from the codon for the family conserved proline on helix VI. However, as is the case for CXCR4, where the 3IL loop is small, cDNA for the fusion protein is inserted directly into the 3IL loop position dictated by spacing from helix V with no resulting excision of intervening nucleotides. Specifically, T4L is inserted into CXCR4 based on spacing between a conserved proline on helix V and a C-terminal truncation was generated based on literature precedence. Each synthesized cDNA is flanked by an out of frame AscI (GGCGCGCCG) restriction site on the 5′ end and an in frame FseI (GGCCGGCC) on the 3′ end for sub-cloning into a set of four expression vectors. Viral DNA is then generated, amplified from these vectors according to standard protocols and titered using flow cytometry to measure the population of cells expressing the virally encoded GP64 protein.

Protein Expression of CXCR4-T4L

With titered virus in hand, small scale expression trials are carried out in a volume of 5 mL/experiment. Expression levels are assessed using flow cytometry to measure the mean fluorescence intensity (MFI) and percentage of cells expressing the FLAG epitope encoded by the expression screening vectors. Expressing cells are tested with and without permeabilization to generate a ratio between protein inserted in the plasma membrane and protein inside the cellular trafficking machinery. A correlation between cell surface expression and overall protein expression is demonstrated, as well as a correlation between stability and the ratio of cell surface expression/total expression. In addition to these assays, small scale purification after solubilization with dodecyl maltoside (DDM) is carried out to determine the quantity of recoverable protein as well as the quality as measured by size exclusion chromatography. Based on these data it was apparent that the T4L fused receptor is expressing and that it is dimerizing in a ligand independent manner, an indicator of C-terminal non-specific interactions in other receptors. Thus, a C-terminal truncation mutant of CXCR4 is generated.

Protein Purification

The C-terminal truncation of T4L fused CXCR4 was scaled up to production sized expression (5-10L of cell culture) and further processed by large scale purification efforts intended for crystallization trials. Briefly 5-10 L of cells culture are centrifuged and washed with PBS followed by freezing at −80° C. The frozen cellular material is then resuspended in 820 mL of lysis buffer (10 mM Hepes pH 7.5, 10 mM MgCl2, 20 mM KCl) supplemented with protease inhibitor (Roche). The cell suspension is lysed by 20 strokes of a dounce homogenizer and centrifuged at 45,000 rpm in Ti45 ultracentrifuge for 30 minutes. The resulted pellet was separated from the supernatant, resuspended and the process repeated six times to ensure complete removal of soluble protein material. On the final resuspension step the membranes were resuspended in 100 mL of lysis buffer containing 40% v/v glycerol, homogenized with 20 strokes of a dounce homogenizer and frozen in 10 mL aliquots at −80° C. for indefinite storage.

For solubilization and purification, each 10 mL aliquot of frozen membranes is resuspended to 25 mL using lysis buffer to which 100 uM AMD070 ligand is added in addition to protease inhibitor at 2× working concentration and 2 mg/mL iodoacetamide. The membranes are allowed to thaw and incubate with ligand at an appropriate temperature for at least 30 minutes. After the incubation the mixture is diluted two-fold with a 2× solubilization buffer containing 100 mM Hepes pH 7.5, 1M NaCl, 2% w/v DDM 0.2% w/v CHS. The solubilization is allowed to proceed with agitation for at least 2 hours at 4° C. after which insoluble material is separated by centrifugation and discarded. The supernatant is isolated and allowed to bind to 0.5 mL of Talon (Clontech) IMAC resin charged with Co2+ in the presence of 20 mM imidizole buffered to 7.5 and 800 mM NaCl. Binding to the Talon IMAC resin is allowed to proceed with agitation at least 4 hours but most commonly overnight. After binding, the slurry is poured into a gravity column and the resin is separated from the supernatant. The resin is then washed with 80 column volumes (CV) of wash buffer (50 mM Hepes pH 7.5, 800 mM NaCl, 20 mM Imidizole, 0.1% w/v DDM, 0.01% w/v CHS and 100 uM AMD070 (or receptor appropriate ligand). After the initial wash the resin is further treated to adjust the NaCl concentration to 500 mM and to increase the ligand concentration to 300 uM. The protein is then eluted from the resin using 200 mM Imidazole and concentrated to 2.5 mL for removal of the excess imidizole with a PD10 desalting column (GE Biosciences). The ligand concentration is increased to 500 uM and the protein is bound to 100 uL of Ni-Sepharose IMAC resin in the presence of 20,000 units of PNGase (NEB) an endoglycosidase capable of removing N-linked glycosylation. The protein is allowed to bind to the resin and deglycosylate for 6 hours after which the resin is washed with imidizole free elution buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 0.05% w/v DDM, 0.01% w/v CHS and 1 mM AMD 070). After the washing step the protein was eluted from the resin using the same buffer but including 200 mM imidizole. After elution the protein is normally concentrated to approximately 50 mg/mL and tested for integrity by SEC. Crystallizable protein should be >90% free of heterogeneity as judged by SDS-PAGE and contain no detectable aggregated species at high protein concentrations as judged by SEC. If the protein remains of high quality it is reconstituted into lipidic cubic phase containing cholesterol. The reconstituted protein is then dispensed onto glass sandwich crystallization plates and tested for crystallization using the screening methodology described in this Example and Example 1. After mixing, the protein-laden lipidid cubic phase mixture will comprise 3.6-7.2% w/w sterol, 56.5-52.8 w/w % Monoolein and 40% w/w protein solution (a 3:2 ratio of lipid mixture to protein). Initial crystallization conditions use PEG 400 between 25-35%, a salt between 50-500 mM, and a pH between 5.0-7.5.

Example 3

High Resolution Crystal Structure of a Human β2-Adrenergic G Protein-Coupled Receptor T4 Lysozyme Fusion Protein

The engineering, functional properties, expression and purification of crystallization grade β2AR-T4L protein are described in more detail in Example 4. Briefly, β2AR-T4L was generated by three distinct modifications to β2AR: (1) a fusion protein was created by replacement of the third intracellular loop with T4L, (2) the carboxyl terminal 48 amino acids were deleted, and (3) a glycosylation site at Asn187 was eliminated through a glutamate substitution. β2AR-T4L was expressed in Sf9 insect cells, solubilized in 1% dodecylmaltoside, and purified by sequential antibody and ligand affinity chromatography. Using the modified lipidic cubic phase (LCP) crystallization procedure described herein, wherein crystals are grown from a cholesterol-doped monoolein cubic phase, β2AR-T4L crystals were obtained that diffract to a resolution of 2.2 Å. The structure was solved at 2.4 Å resolution. Compared to crystallization in detergents, LCP provides a more native, lipid environment for crystallization, as well as a confinement of protein molecules to two-dimensional membrane sheets that may facilitate the crystallization process through the formation of Type I packing interactions (Caffrey, Curr Opin Struct Biol 10, 486 (2000); Deisenhofer, EMBO J 8, 2149 (1989); Landau et al., Proc Natl Acad Sci U S A 93, 14532 (1996)).

Methods

Lipidic Cubic Phase Crystallization.

Crystals of engineered human β2AR 032AR-T4L) grown from bicelles could not be optimized beyond 3.5 Å resolution (FIG. 5). Lipidic cubic phase (LCP) crystallization trials were therefore performed using an in meso crystallization robot (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004)). 96-well glass sandwich plates (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov, et al, J Membr Biol 195, 165 (2003)) were filled with 25 or 50 nL protein-laden LCP drops overlaid by 0.8 μL of precipitant solution in each well and sealed with a glass coverslip. All operations starting from mixing lipid and protein were performed at room temperature (˜21-23° C.). Crystals were obtained in 30-35% (v/v) PEG 400, 0.1-0.2 M sodium sulfate, 0.1 M Bis-tris propane pH 6.5-7.0 and 5-7% (v/v) 1,4-butanediol using 8-10% (w/w) cholesterol in monoolein as the host lipid (FIG. 6A). Addition of cholesterol and 1,4-butanediol dramatically improved crystals size and shape, thereby enabling high-resolution diffraction. In this instance, additions of phospholipids (dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, asolectin) to the main host LCP lipid monoolein (either alone or in combination with cholesterol) failed to improve crystal quality.

Crystal Harvesting

The average size of the harvested crystals was 30×15×5 μm (largest crystal was 40×20×7 μm). Crystals were harvested directly from the glass sandwich plates, even though these plates have been specifically designed for screening and optimization (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov, et al, J Membr Biol 195, 165 (2003)). Crystals were scooped directly from the LCP using 30 or 50 μm aperture MiTeGen MicroMounts and plunged into liquid nitrogen. Care was taken to drag as little as possible lipid around the crystal to decrease unwanted background scattering. Attempts to dissolve the lipids, either by increasing concentration of PEG 400 or using a mineral oil, typically resulted in a decrease in diffraction power of the crystals.

Data Collection

X-ray data were collected on the 231D-B beamline (GM/CA CAT) at the Advanced Photon Source, Argonne, IL using a 10 μm minibeam (wavelength 1.0332 Å) and a MarMosaic 300 CCD detector (FIG. 6B). Several complete datasets were collected from single crystals at resolution between 2.8 and 3.5 Å using 5× attenuated beam, 5 s exposure and 1° oscillation per frame. However, some crystals diffracted to a maximum of 2.2 Å resolution upon 5 s exposure with 1× attenuated beam. Therefore, 10-20° wedges of high-resolution data were collected from more than 40 crystals (some of the crystals were large enough to allow 2-3 translations). 31 of the best datasets from 27 independent crystals were then combined and scaled against the lower resolution full dataset to obtain complete 2.4 Å data.

One of the challenges during data collection was visualization of colorless microcrystals within an opaque frozen lipid phase and aligning them with the 10 μm minibeam. Because the crystals could not be adequately visualized through the inline optics at the beamline, alignment-by-diffraction techniques were employed. The present invention provides, in one aspect, an optimized crystal search algorithm to locate the crystals without the minibeam. First, the area of the loop containing lipid was scanned in the vertical direction with a highly attenuated and slitted 100×25 μm beam. When diffraction was found, the crystal location was further confined by two additional exposures to an area of ˜50×25 μm. This area was further coarse-scanned with the collimated and 10× attenuated minibeam using 15 μm steps, following by fine-tuning the position using 5 and 2 μm steps. After locating the crystal in one orientation the loop was rotated 90° and the procedure was repeated. Typically during alignment the crystal was exposed ˜10 times using 10× attenuated beam and 2 s exposures.

Data Processing

A 90% complete, 2-fold redundant monoclinic dataset was processed from one crystal diffracting to 2.8 Å resolution. Initial indexing of lattice parameters in spacegroup C2 and crystal orientation were performed using HKL2000 (Otwinowski, et al, in Methods in Enzymology C. W. J. Carter, R. M. Sweet, Eds. (Academic Press, New York, 1997), vol. 276, pp. 307-326). The refined lattice parameters and space group were implemented in the data processing program XDS for spot integration which models error explicitly for radiation decay, absorption, and rotation (Kabsch, J Appl Crystallogr 26, 795 (1993)). Because data was collected using a 10 μm beam from microcrystals, maintaining the crystal orientation at the beam center during data collection was especially problematic. It appeared that XDS modeled the crystal orientation error upon rotation about the phi axis better than other data processing programs that were tried, resulting in better merging statistics. In addition to rotational error, the radiation decay was also an issue that was partially corrected by the XDS processing program, enabling a more reliable scaling of datasets from different crystals and translations of crystals. The 2.8 Å data was used as a scaling reference for incorporation of additional wedges of data collected at a much higher exposure. Each new dataset was indexed in XDS using the original unit cell parameters as constants which were then refined along with the crystal orientation, beam geometry, and mosaicity parameters. The refinement was generally stable, resulting in very similar unit cell constants which enabled subsequent scaling. All of the integrated wedges of data were then tested individually against the scaling reference set and included in the final scaled dataset if the merging statistics remained acceptable upon incorporation of the data. In total, 31 wedges of data from 27 crystals were combined with the scaling reference dataset, 22 of which diffracted to a resolution of 2.4 Å or better. Each of the higher resolution datasets were exposed to a much larger dose of radiation resulting in a rapid decay in intensity. Typically 10°-20° wedges were collected from each crystal or translation, 5°-7° of which had diffraction data to 2.4 Å. The final merging statistics for the dataset are shown in Table 2. Based on the mean F/σ(F) of reflections near the three crystallographic axes, the effective resolution is estimated to be 2.4 Å along b* and c* and 2.7 Å along a*. The anisotropy results in the high merging R factors in the last few resolution shells despite the significant I/σ(I) values. The anisotropy is either an inherent property of the crystals or the result of a preferential orientation of the crystals within the mounting loop. Thus, the higher resolution shells were filled in anisotropically by incorporation of the additional data at high exposure levels, while the lower resolution shells have a very high redundancy and low anisotropy.

TABLE 2
Data processing statistics from XDS. A comparison is made between data filtered by a sigma cutoff
and the complete set. All of the data was used in structure solution and refinement.
# Observed# UniqueI/
ResolutionreflreflRedundancyCompletenessRsymSIGMAR-measRmrgd-F
Signal/noise ≧ −3
10  335233310.187.40%6.60%23.256.90%2.70%
8  359135410.199.40%7.60%22.68.00%2.80%
6  10480100310.499.60%9.90%20.0110.50%4.00%
3  1260081196810.599.80%13.30%14.0114.00%6.60%
2.833158313010.6100.10%38.00%6.4239.90%18.70%
2.719702189310.499.70%49.50%4.9652.10%24.90%
2.623772227510.499.90%60.20%4.0763.40%30.10%
2.51410825585.599.30%58.90%2.6965.10%51.80%
2.41467230604.899.10%67.80%2.1875.70%62.80%
Total248843265749.499.50%12.70%9.6213.40%11.40%
Signal/noise ≧ 0
10  335233310.187.40%6.60%23.256.90%2.70%
8  359135410.199.40%7.60%22.68.00%2.80%
6  10480100310.499.60%9.90%20.0110.50%4.00%
3  1256281192310.599.40%13.30%14.0614.00%6.50%
2.832679307710.698.40%37.60%6.5439.50%17.70%
2.719346184910.597.40%48.80%5.0851.30%23.40%
2.623201221010.597.00%58.90%4.262.00%28.00%
2.51346124065.693.40%56.40%2.8862.30%45.90%
2.41383328274.991.50%64.10%2.471.50%54.30%
Total245571259829.597.30%12.60%9.8513.30%10.80%
Rsym = Σhkl|I(hkl) − <I(hkl)>|/Σhkl(hkl), where <I(hkl)> is the mean of the symmetry equivalent reflections of I(hkl);
R-meas = redundancy independent Rsym;
Rmrgd-F = indication of amplitude quality. See, e.g., Murshudov, et al, Acta Crystallogr D D53, 240 (1997).

Structure Solution and Refinement

Initial phases for β2AR-T4L were obtained by molecular replacement using both T4-lysozyme (PDB ID Code 2LZM) and a polyalanine model of the rhodopsin seven-transmembrane bundle (PDB ID Code 1U19) as search models. It was necessary to trim the lysozyme search model to remove residues 12-71 as that domain had shifted conformations relative to the larger section. This domain was later reintroduced to the model by fitting into observed density. Molecular replacement was carried out using the program Phaser by first placing the truncated lysozyme (RFZ=3.74; TFZ=3.65) followed by the rhodopsin model (RFZ=5.2; TFZ=7) (McCoy, Acta Crystallogr D Biol Crystallogr 63, 32 (2007)). In order to optimize placement of the receptor each of the seven helices was refined independently by rigid body maximum likelihood refinement as implemented in Refmac (Initial Rwork/Rfree=0.50/0.51) (Murshudov, et al, Acta Crystallogr D D53, 240 (1997)).

Initial rounds of refinement were carried out using restrained parameters in Refmac. Model rebuilding was performed in Coot utilizing 2 Fo-Fc sigma-A weighted maps, as well as density modified maps calculated using Resolve prime-and-switch phasing which reduces model bias introduced by model based phasing methods (Terwilliger, Acta Crystallogr D D56, 965 (2000)). The Resolve calculated maps were superior to the sigma-A weighted ones in that more of the main chain density could be traced. Density for the bound ligand was visible early in the refinement but was not modeled immediately to allow an unbiased assessment of the phase quality through the improvement of the signal/noise of the observed ligand density. The structure quality is excellent (Table 3), with strong electron density in particular observed in the ligand binding site (FIG. 7), cholesterol binding sites (FIG. 8A), and the proline helix kinks (FIG. 8B).

TABLE 3
Data collection and refinement statistics
β2AR-T4L
Data collection (APS GM/CA CAT 23ID-B, 10 μm beam)*
Space groupC2
Cell dimensions
a, b, c (Å)106.32, 169.24, 40.15
β (°)105.62
No. of reflections processed245,571
No. unique reflections26,574
Resolution (Å)50-2.4(2.5-2.4)
Rsym12.7(67.8)
Mean I/σ(I)9.6(2.2)
Completeness (%)99.5(99.1)
Redundancy9.4(4.8)
Refinement*
Resolution (Å)20-2.4(2.46-2.4)
No. reflections (test set)25,247(1,310)
Rwork/Rfree19.8 (27.0)/23.2 (30.1)
No. atoms3,805
Protein3,544
Ions, lipids, ligand and other213
Water48
Overall B-values (Å2)82
β2AR77
T4-Lysozyme75
Carazolol55
Lipid100
R.m.s deviations
Bond lengths (Å)0.013
Bond angles (°)1.5
Ramachandran plot statistics (%)
(excl. Gly, Pro):
Most favored regions94.8
Additionally allowed regions5.0
Generously allowed regions0.2
Disallowed regions0
*Highest resolution shell is shown in parenthesis. Rsym = Σhkl|I(hkl) − <I(hkl)>|/Σhkl(hkl), where <I(hkl)> is the mean of the symmetry equivalent reflections of I(hkl).

Analysis of β2A Receptor Topology

The final model of β2AR-T4L includes 442 amino acids. β2AR-T4L was treated with iodoacetamide during purification to eliminate free thiols. The model includes a palmitic acid covalently bound to Cys341 (GPCRs are frequently post-translationally modified with palmitoylate on cysteine residues at the C-terminal tail) and an acetamide molecule bound to Cys2656.27. Throughout the description, residues are designated by their position within the β2AR sequence and their Ballesteros-Weinstein designation as a superscript where applicable. Ballesteros-Weinstein numbering is used throughout the text as superscripts to the protein numbering. Within each helix is a single most conserved residue among the class A GPCRs. This residue is designated x.50 where x is the number of the transmembrane helix. All other residues on that helix are numbered relative to this conserved position. The model also includes one carazolol molecule, three cholesterol molecules, two sulfate ions and two butanediol molecules that interact with β2AR. There are also four sulfate ions, a putative disaccharide (modeled as maltose) and a molecule of PEG 400 bound to T4L. For β2AR, excellent electron density is observed for residues 29-342, including the ligand carazolol and the two disulfide bonds Cys1063.25-Cys1915.36 and Cys1844.76-Cys1905.29. The palmitic acid at Cys341 is clearly visible in Fo-Fc omit maps; however, the quality of the electron density is lower than for the rest of the receptor. The N-terminus (residues 1 to 28) and the majority of the C-terminus (residues 343 to 365) are disordered and not visible in the structure.

The β2AR has a fold composed of seven transmembrane helices forming a helical bundle (FIG. 9A). The residues that make up the helices (I to VII) in β2AR are as follows: helix 1291.28 to 601.59, helix II 672.38 to 962.67, helix III 1033.22 to 1363.55, helix IV 1474.39 to 1714.63, helix V 1975.36 to 2295.68, helix VI 2676.29 to 2986.60, and helix VII 3057.32 to 3287.55. The residues forming the intracellular loops (ICL) and extracellular loops (ECL) of β2AR are: ICL1 611.66 to 66237, ECL1 972.68 to 1023.21, ICL2 1373.56 to 1464.38, ECL2 1724.64 to 1965.35, ICL3 2305.69 to 2666.28 (residues 231 to 262 are replaced by T4-lysozyme residues 2 to 161), and ECL3 2996.61 to 3047.31. Helices II, V, VI and VII each have a proline-induced kink at conserved positions along the span of the transmembrane segments. These kinks are thought to enable the structural rearrangements required for activation of G protein effectors (Yohannan et al., Proc Natl Acad Sci U S A 101, 959 (2004)). In addition to the seven membrane spanning helices, β2AR possesses two other helical segments: helix VIII, which is believed to be common to all rhodopsin-like GPCRs (Katragadda et al., Biochim Biophys Acta 1663, 74 (2004)), and an unexpected, short helical segment in the middle of ECL2, which is not present in rhodopsin, and was not predicted by computational secondary structure analysis (FIG. 9A).

In the β2AR-T4L construct, T4L is fused to the truncated cytoplasmic ends of helices V and VI. In the crystal structure, the T4L moiety is tilted slightly away from the center axis of β2AR drawn normal to the membrane (FIG. 9B). As a result, interactions between T4L and β2AR are minimal, with only 400A2 of surface area buried between them. The intramolecular contacts between T4L and β2AR include salt bridges between the side chains of T4L-Asp159 and the side-chain amine of β2AR-Lys2275.66 (distance 3.4 Å) and between the guanidinium group of T4L-Arg8 with the side-chain carboxyl of β2AR-Glu2686.30 on helix VI (distance 3.2 Å) (FIG. 9C, Table 4). The latter interaction is noteworthy because it differs from rhodopsin where Glu6.30 forms an ionic bond with Arg3.50 of the conserved D(E)RY motif. This interaction is postulated to be important for maintaining rhodopsin in the inactive state, but the charged groups of the two residues [Arg1313.50 (NH1) and Glu2686.30 (0E1)] are 10 Å apart in the β2AR-T4L structure. The remainder of the lysozyme molecule provides important crystal packing interactions, but does not appear to influence significantly the receptor structure.

TABLE 4
Direct contacts between β2AR and T4L
van der Waals Contacts
β2AR atomT4 Lysozyme
Leu2305.69Trp 158
Lys2636.25Asp159
Cys2656.27Ile9
Leu2666.28Ile9
Leu2666.28Glu5
Hydrogen Bond and Salt Bridge Contacts
β2AR atomT4 LysozymeDistance (Å)
Lys2275.66 (NZ)Asp159 (OD1)3.4
Gln2295.68 (O)Asn2 (N)3.1
Gln2295.68 (O)Asn2 (ND2)3.2
Gln2686.30 (OE2)Arg8 (NH2)3.2
Covalent bonds
β2AR atomT4 Lysozyme
Leu2305.69Asn2
Lys2636.25Tyr161

Crystal Packing Interactions

The β2AR-T4L protein is packed in a C-centered monoclinic lattice with one molecule per asymmetric unit (FIG. 10A). Membrane protein generally can form two types of crystal packing: Type I represents stacks of two dimensional crystals ordered in the third dimension via interactions of hydrophilic parts of membrane proteins. Type II crystals are composed of membrane proteins whose hydrophobic part is shielded by a detergent micelle and all crystal contacts are formed through hydrophilic, solvent exposed parts of protein molecules. As observed in all previous lipidic mesophase grown crystals (Schwartz, et al., Annu Rev Pharmacol Toxicol 46, 481 (2006)), the β2AR-T4L crystals adopt Type I packing, featuring a multilayered arrangement in accordance with proposed crystallization mechanism (Caffrey, Curr Opin Struct Biol 10, 486 (2000); P. Nollert, et al., FEBS Lett 504, 179 (2001)). Within each layer, protein molecules form arrays of parallel, symmetry-related dimers. There are four distinct crystal-packing interactions within each layer, three of which are mediated by T4L. The fourth interaction in the array is between two receptor molecules related by a crystallographic two-fold rotation axis. This is the sole interaction between symmetry-related receptors, and is mediated primarily by ordered lipids consisting of six cholesterol and two palmitic acid molecules, the latter being covalently attached to Cys341 in the C-terminal portion of the receptor (O'Dowd et al., J Biol Chem 264, 7564 (1989)) (FIG. 10B). These eight lipid molecules form a two-fold symmetric sheet between receptors. The only direct receptor-receptor contact involves a 2.7 Å pair of ionic interactions between the charged amine group of Lys601.59 in helix I and the carboxylate of Glu338 in helix VIII from the symmetry-related receptor. Remarkably, of the 515 Å2 buried at the receptor symmetry interface, 73% of the crystal contact surface area is mediated by ordered lipid, while only 27% is contributed by protein-protein contacts. The stacking interactions between layers are formed between T4L and extracellular loops ECL2 and ECL3 of the receptor (FIG. 10A). It is unlikely that these contacts affect the orientation of these loops due to the small size of ECL3 and the rigid architecture of ECL2.

Lipid Mediated Receptor Association

Many GPCRs including β2AR are thought to exist as dimers in the plasma membrane, although the location of the dimer interface and the functional significance of dimerization is not clear (Milligan, Mol Pharmacol 66, 1 (2004)). The observation of ordered lipids in the helix I and VIII interface between two symmetry related molecules suggests the association is physiologically relevant (Angers, et al., Proc Natl Acad Sci U S A 97, 3684 (2000); Javitch, Mol Pharmacol 66, 1077 (2004); Mercier, et al., J Biol Chem 277, 44925 (2002)). Associations between the equivalent regions of rhodopsin have been found in crystal structures (Salom et al., Proc Natl Acad Sci U S A 103, 16123 (2006); Schertler, Curr Opin Struct Biol 15, 408 (2005)) (FIG. 10C).

The role of cholesterol in the physiologic function of β2AR is well documented. Depletion of cholesterol from the membranes of neonatal cardiac myocytes alters the signaling behavior of endogenous β2AR (Xiang, et al., J Biol Chem 277, 34280 (2002)). In untreated cells, activation of β2AR results in sequential coupling to the G proteins Gs and Gi, producing a biphasic effect on myocyte contraction rate. Upon depletion of cholesterol, the β2AR couples more strongly to Gs. This effect may be due to a role of cholesterol in regulating interactions between the β2AR and G proteins, or an effect of cholesterol on β2AR dimerization. The β2AR couples efficiently to Gs as a monomer (Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), so cholesterol mediated association (dimerization) may reduce the efficiency of β2AR coupling to Gs. The effects of cholesterol depletion on β2AR signaling may also be a secondary effect of altering subcellular signaling compartments. There is evidence that cells may concentrate signaling molecules, such as GPCRs and their cognate G proteins, by way of membrane microdomains or compartments, such as caveolae (Ostrom, et al., Br J Pharmacol 143, 235 (September, 2004)). This compartmentalization may be a major regulator of receptor-effector coupling. Thus, the importance of cholesterol in forming the observed crystallographic association is consistent with its role in β2AR signaling.

Electrostatic Charge Distribution

Electrostatic charge distribution was calculated using APBS (Gether, Endocr Rev 21, 90 (2000)) and mapped onto a molecular surface representation of β2AR. The analysis reveals three polarized areas within the molecule (FIG. 11A). First, the cytoplasmic face of the receptor is involved in G protein interaction and carries a net positive charge even in the absence of ICL3, which also has a predicted overall positive charge (FIG. 11B). The second site is an electrostatically negative region located within the membrane between helices III, IV and V potentially exposed to the lipid alkyl chains, which is unexpected as the burial of charge within the plasma membrane is thermodynamically unfavorable. A glutamate residue at position 1223.41 may partially account for the observed charge distribution. Finally, the binding site cleft is negatively charged and exposed to solvent by an unusual ECL2 architecture and lack of N-terminal interactions. This negative charge may facilitate ligand binding through electrostatic funneling of positively charged catecholamines (FIG. 11B).

Extracellular Region

The ECLs and amino termini of GPCRs, together with the extracellular halves of the transmembrane helices, are believed to define the ligand-binding site of each receptor (Angers et al., Proc Natl Acad Sci U S A 97, 3684 (2000)). Therefore, the ECLs play an important role in the overall pharmacology of any particular receptor. In general, small molecule ligands are thought to bind deeper within the space created by the transmembrane domain helices, whereas larger ligands such as peptides bind closer to the membrane surface near the ECLs (Ji, et al., J Biol Chem 273, 17299 (1998); Gether, Endocr Rev 21, 90 (2000)). Mutagenesis studies suggest that the β2AR binds its ligand deep within the transmembrane helix bundle, which may be related to the observation that the extracellular regions have a rather simple structure with short loops connecting transmembrane helices II and III, and VI and VII (FIG. 12A). ECL2, which links helices IV and V, has a somewhat more extensive architecture that is unanticipated. In contrast to the buried, β-sheet structure of this loop in rhodopsin (FIG. 12B), ECL2 in β2AR is more exposed to the solvent and contains an extra helical segment. Additionally, there is an intra-loop disulfide bond between Cys1844.76 and Cys1905.29 that may help stabilize the more exposed ECL2. A second disulfide bond between Cys1915.30 and Cys1063.25 in helix III effectively ties ECL2 to the transmembrane core (Noda, et al., J Biol Chem 269, 6743 (1994)). The distal portion of ECL2 makes close contacts with ECL1 and contains a glycosylation site at Asn1875.26 (Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), which may serve to mask a grouping of aromatic residues on ECL1; in this construct, Asn1875.26 has been mutated to glutamate to aid in crystallization.

Electron density corresponding to the N-terminus was not apparent in the maps and, therefore, residues 1-28 are not included in the model. This disorder contrasts with rhodopsin, in which the N-terminus interacts extensively with the ECLs, forming a small four-strand β-sheet in conjunction with ECL2. This sheet structure forms a cap that effectively isolates the retinal binding site in a hydrophobic pocket (FIG. 13B). The lack of interactions between the N-terminus of β2AR and ECL2 further enables diffusible ligand access to the binding site. However a completely disordered N-terminus may be an artifact induced by the presence of the N-terminal Flag tag which carries an overall positive charge and may disrupt N-terminal interactions.

The short helical region on ECL2 adds a rigid structural element that, along with the two disulfide bonds, constrains the loop to a small range of conformations and helps stabilize the receptor by linking three transmembrane helices (FIG. 13A). This rigid conformation may help to stabilize the core of the receptor and lock ECL2 in a conformation that does not hinder access to the binding pocket.

Ligand Binding Site and Comparison to Rhodopsin

Carazolol is a partial inverse agonist that binds with picomolar affinity to β2AR-T4L producing a reduction of the basal activity of the receptor. The crystal structure reveals extensive interactions between the receptor and carazolol that position the carbazole moiety adjacent to Phe2896.51, Phe2906.52, and Trp2866.48 (FIG. 13A, FIG. 7, and Table 5). In contrast, cis-retinal is a full inverse agonist covalently bound to rhodopsin, which suppresses all activity towards transducin (Palczewski, Annu Rev Biochem 75, 743 (2006)). Carazolol and retinal occupy similar spaces in their respective receptors, with significant overlap of the non-aromatic regions of carazolol. However, the β-ionone ring of retinal extends deep into the binding pocket of rhodopsin and contacts residues on helix V and VI, where it is sandwiched between Phe2125.47 and Tyr2686.51, and interacts with the highly conserved Trp2656.48 (FIG. 13B). It has been proposed that changes in the rotamer of Trp2656.48 occur upon activation of rhodopsin and related family members, and constitutes the “toggle switch” for receptor activation (Schwartz, et al., Annu Rev Pharmacol Toxicol 46, 481 (2006)). Accordingly, the interactions between c/s-retinal and Trp2656.48 are likely to contribute to the absence of basal activity in rhodopsin. Carazolol does not interact directly with the toggle switch on helix VI, however it lowers the basal activity of the receptor, and may do so by interacting with Phe2896.51 and Phe2906.52, which form an extended aromatic network surrounding the highly conserved Trp2866.48. As a result, Trp2866.48 adopts the rotamer associated with the inactive state. Thus, the steric constraints imposed by Phe2906.52 appear to structurally mimic the interaction of the β-ionone ring of retinal with the conserved Trp2656.48 and Phe2125.47 on rhodopsin (Shi et al., J Biol Chem 277, 40989 (2002)) (FIG. 13C).

TABLE 5
Direct contacts between β2AR and carazolol.
β2AR atomCarazololDistance (Å)
Hydrogen Bond and Salt Bridge Contacts
Asp1133.32 (OD2)N192.9
Asp1133.32 (OD1)O172.6
Ser2035.42 (OG)N73.2
Asn3127.39 (ND2)O172.9
Asn3127.39 (OD1)N192.9
Tyr3167.43 (OH)N193.4
Hydrophobic and Aromatic Interactions
(closest distance for each residue, <4 Å)
Trp1093.28 (CH2)C213.8
Val1143.33 (CG1)C113.9
Val1173.36 (CG1)C124.0
Thr1183.37 (OG1)C113.9
Phe1935.32 (CE2)C63.5
Tyr1995.38 (CE2)C23.9
Ser2075.46 (CB)C103.6
Trp2866.48 (CH2)O173.4
Phe2896.51 (CE2)O143.7
Phe2906.52 (CZ)C123.5
Asn2936.55 (ND2)C53.6
Tyr3087.35 (OH)C63.6

Structural Alignment and Helix Bundle Reorganization

It has long been thought that class A GPCRs share a similar architecture due to their predicted seven transmembrane helical bundles and sequence conservation within the membrane spanning regions (Lefkowitz, Nat Cell Biol 2, E133 (2000)). We aligned the structure of β2AR-T4L to highest resolution structure of rhodopsin (PDB ID Code 1U19) to evaluate the similarities and differences in ligand binding modes. We used difference distance matrices to select non-divergent areas between the two structures that align to reveal the differences in helix orientation between β2AR-T4L and rhodopsin. For the alignment, residues on β2AR were aligned to equivalent residues on Rhodopsin, respectively: 43-59 to 47-63; 67-95 to 71-99; 122-135 to 126-139; 285-296 to 264-275.

Relative to rhodopsin, the following helical shifts are seen in β2AR-T4L: the extracellular portions of helices I and III angle away from the center of the receptor, helix IV is translated away from the center of the receptor, helix V is translated closer to the center of the receptor and helix VI angles away from the receptor on the cytoplasmic end (FIG. 14). The largest difference is in helix I, which lacks a proline-induced kink found in rhodopsin and is comparatively straight. The angle between the rhodopsin and β2AR positions of helix I is approximately 18° with a shift of 7 Å at the apex on the extracellular face. This structural difference may arise from the need for an accessible binding site in β2AR, which is provided in part by a lack of interactions between the N-terminus and extracellular loop segments. In contrast the N-terminal region in rhodopsin occludes the retinal-binding site through extensive interactions with the extracellular loops (FIG. 12B). Helix V of β2AR is closer to the binding pocket by approximately 3.5 Å on average and its lumenal end is angled more towards helix VI. Helix IV of β2AR is further from the binding site, possibly to remove steric clashes resulting from the modified position of helix V (FIG. 14B, 14C). Helix III pivots further from the binding site about a fulcrum located close to the cytoplasmic end (FIG. 14C). The angle formed between rhodopsin helix III and the β2AR helix III is approximately 7°, yielding a 4 Å displacement out of the binding pocket at the cytoplasmic end of the helix. Helix VI is positioned further from the center of the receptor at the cytoplasmic end as compared to rhodopsin, which is caused by a slight difference in the angle about the proline-induced kink in the helix (FIG. 14C).

The ligand-binding pocket is formed by both structurally conserved and divergent helices as compared to rhodopsin (FIG. 14D). Helices III and V are two of the most conformationally shifted helices and contain the canonical catecholamine binding residues associated with activation of adrenergic family of receptors (Strader et al., J Biol Chem 263, 10267 (1988); Strader, et al., J Biol Chem 264, 13572 (1989); Liapakis et al., J Biol Chem 275, 37779 (2000)). The comparison with rhodopsin shows that the structurally conserved helices provide a common core present throughout the class A GPCRs, whereas the variable helices confer binding site plasticity with a resulting architecture capable of binding a large spectrum of ligands.

Comparison to Rhodopsin-Based GPCR Models

Since the determination of the inactive dark-state rhodopsin structure (Palczewski et al., Science 289, 739 (2000)), a number of homology models of other class A GPCRs have been reported (Bissantz, et al., Proteins 50, 5 (2003); Fano, et al., J Chem Inf Model 46, 1223 (2006); Hobrath, et al., J Med Chem 49, 4470 (2006); Nowak, et al., J Med Chem 49, 205 (2006); Zhang, et al., PLoS ComputBiol 2, e13 (2006)). Typically, homology models start by alignment of so-called fingerprint motifs that are common among the family. These fingerprint motifs are extrapolated to assign coordinates for the entire helical bundle. Loop regions are either ignored or modeled based on databases of loop conformations depending on the application (Bissantz, et. al, Proteins 50, 5 (2003)). A number of models exist for β2AR, some of which have been improved upon with supporting biochemical data (Bissantz, et. al, Proteins 50, 5 (2003); Zhang, et al., PLoS ComputBiol 2, e13 (2006); Freddolino et al., Proc Natl Acad Sci U S A 101, 2736 (2004); Furse, et al., J Med Chem 46, 4450 (2003); Gouldson et al., Proteins 56, 67 (2004)). When compared to the β2AR structure reported here (according to the methods described above in this Example), however, all of these models were more similar to rhodopsin, as were models for other receptors (e.g. dopamine, muscarinic, and chemokine). This highlights a general shortcoming in homology models generated from a single structural template. The structural divergence between β2AR and rhodopsin would be quite difficult to predict accurately using only rhodopsin as a template.

Example 4

Structural Insights into β2 Adrenergic Receptor Function

Methods

Molecular Biology for Generation of Mammalian and Sf9 Expression Constructs.

The insect cell expression plasmid that was used as a template for modification of the human β2AR gene has been described previously (Yao et al., Nat Chem Biol 2, 417 (2006)): the wild-type coding sequence of the human β2AR (starting at Gly2) was cloned into the pFastbac1 Sf-9 expression vector (Invitrogen) with the HA signal sequence followed by the Flag epitope tag at the amino terminus and the third glycosylation site mutated as N187E. Using this template, a TAA stop codon was placed between Gly365 and Tyr366, terminating translation without the 48 C-terminal residues of the wild-type β2AR (β2AR365″). A synthetic DNA cassette encoding the T4 Lysozyme (WT*-C54T, C97A) protein was made by overlapping extension PCR of 50-base oligonucleotides. This cassette was amplified and inserted into the β2AR365 construct between Ile2335.72 and Arg2606.22 (FIG. 21A), using the Quickchange Multi protocol (Stratagene). The corresponding mammalian cell expression plasmid was made by amplifying the entire fusion gene and cloning it into pcDNA3 (Invitrogen). Further deletions in the Sf9 and mammalian cell constructs were made using appropriate synthetic oligonucleotides in the Quickchange Multi protocol (Stratagene). All constructs were confirmed by sequencing.

HEK293 Cell Staining and Immunofluorescence Staining.

HEK293 cells were cultured on plastic dishes at 37° C. with 5% CO2 in Dulbecco's modified Eagle's medium (Cellgro) with 5% fetal bovine serum. For an individual expression experiment, cells at confluency were split, and approximately 100,000 cells were used to seed glass cover slips in the same medium. After 2 d, cells were transfected with the addition of 1 μg of a given pcDNA3-receptor plasmid and 3 μl of Fugene 6 reagent (Roche). 48 h after transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, blocked with PBS+2% goat serum, permeabilized with PBS+2% goat serum+0.5% Nonidet P-40 (Sigma), stained with Alexa488-conjugated M1 anti-FLAG antibody (for receptor) plus DAPI (nuclear) in blocking buffer, and washed with blocking buffer. Cover slips were mounted on microscope slides with Vectashield (Vector Labs) and dried overnight. Staining was visualized with an Axioplan 2 fluorescence imaging system, using a 63× objective and either green (Alexa488/FITC) or blue (DAPI/Hoechst) filter sets. A plasmid pcDNA3-β1AR, expressing an N-terminal FLAG-tagged β1 adrenergic receptor, was used as a positive control for cell-surface staining. Empty pcDNA3 was used as a negative control to assess background staining.

Expression and Purification of β2AR-T4L from Baculovirus-Infected Sf9 Cells.

Recombinant baculovirus was made from pFastbac1-β2AR-T4L using the Bac-to-Bac system (Invitrogen), as described previously (Yao et al., Nat Chem Biol 2, 417 (2006)). The β2AR-T4L protein was expressed in Sf9 insect cells infected with this baculovirus, and solubilized according to previously described methods (Kobilka, Anal Biochem 231, 269 (1995)). Dodecylmaltoside-solubilized receptor with the N-terminal FLAG epitope (DYKDDDA) (SEQ ID NO: 1) was purified by M1 antibody affinity chromatography (Sigma), treated with TCEP/iodoacetamide, and further purified by alprenolol-Sepharose chromatography (Kobilka, Anal Biochem 231, 269 (1995)) to isolate only functional GPCR. Eluted alprenolol-bound receptor was re-bound to M1 FLAG resin, and ligand exchange with 30 μM carazolol was performed on the column. β2AR-T4L was eluted from this final column with 0.2 mg/ml FLAG peptide in HLS buffer (0.1% dodecylmaltoside, 20 mM Hepes, 100 mM NaCl, pH 7.5) plus 30 μM carazolol and 5 mM EDTA. N-linked glycolsylations were removed by treatment with PNGaseF (NEB). Protein was concentrated from ˜5 mg/ml to 50 mg/ml with a 100 kDa molecular weight cut-off Vivaspin concentrator (Vivascience), and dialyzed against HLS buffer plus 10 μM carazolol.

Binding Measurements on Wild-Type β2AR and β2AR-T4L from Membranes.

Membrane preparation from baculovirus-infected Sf9 cells was performed as described previously (Swaminath, et al., Mol Pharmacol 61, 65 (2002)). For each binding reaction, membranes containing 0.7 μg total membrane protein were used. Saturation binding of [3H]-dihydroalprenolol (DHA) was measured by incubating membranes resuspended in 500 μl binding buffer (75 mM Tris, 12.5 mM MgCl2, 1 mM EDTA, pH 7.4, supplemented with 0.4 mg/ml BSA) with 12 different concentrations of [3H]DHA (Perkin Elmer) between 20 pM and 10 nM. After 1 h incubation with shaking at 230 rpm, membranes were filtered from the binding reactions with a Brandel harvester, washed with binding buffer, and measured for bound [3H]DHA with a Beckman LS6000 scintillation counter. Non-specific binding was assessed by performing identical reactions in the presence of 1 μM alprenolol. For competition binding, membranes resuspended in 500 μl binding buffer were incubated with 0.5 nM [3H]DHA plus increasing concentrations of the competing ligand (all compounds were purchased from Sigma). For (−)-isoproterenol and (−)-epinephrine, concentrations were 100 pM-1 mM, each increasing by a factor of 10. For salbutamol, concentrations were 1 nM-10 mM. For ICI-118,551 and formoterol, concentrations were 1 pM-10 μM. Non-specific binding was measured by using 1 μM unlabeled alprenolol as competing ligand. Each data point in the curves in FIGS. 2A and S1 represents the mean of three separate experiments, each done in triplicate. Binding data were analyzed by nonlinear regression analysis using Graphpad Prism. The values for Kd of [3H]DHA and Ki of other ligands are shown in Table 6.

TABLE 6
Saturation Binding
[3H]DHAKd ± SE (nM)Bmax (pmol/mg)
β2AR0.161 ± 0.01230.0 ± 0.5
β2AR-T4L0.180 ± 0.01621.6 ± 0.5
Competition Binding
Ki [S.E. interval]Ki [S.E. interval]
Ligandfor β2AR (nM)for β2AR-T4L (nM)
(−)-isoproteronol50.6 [48.9-52.3]15.7 [15.2-16.2]
(−)-epinephrine175 [163-188] 56.0 [52.8-59.4]
salbutamol728 [708-750] 307 [291-323] 
ICI-118,551 0.617 [0.570-0.668] 0.626 [0.591-0.662]
formoterol3.60 [3.39-3.83]1.68 [1.55-1.81]
Binding affinities of different ligands for the wild-type β2AR and the fusion protein β2AR-T4L.
The saturation and competition binding curves shown in FIG. 22 were fit to theoretical saturation and one-site competition binding models, using the program Graphpad Prism.
Ki values were calculated using the Cheng-Prusoff equation: Ki = IC50/(1 + [ligand]/Kd).

Bimane Fluorescence Experiments on Purified, Detergent-Solubilized Receptors

β2AR-T4L and β2AR365 were purified as described above, with two important differences. First, prior to iodoacetamide treatment, FLAG-pure receptor at 2.5 μM (measured by soluble [3H]DHA binding) was incubated with 5 μM monobromobimane for 1 h at 4° C. Second, after binding the bimane-labeled alprenolol-Sepharose-purified receptor to M1 antibody resin, the column was washed extensively with ligand-free buffer before elution. Based on previous precedent (Ghanouni, et al., Proc Natl Acad Sci U S A 98, 5997 (2001)), this protocol is expected to target primarily Cys2656.27 for fluorophore derivitization. Fluorescence spectroscopy was performed on a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon Inc.) with photon-counting mode, using an excitation and emission bandpass of 5 nm. All experiments were done at 25° C. For emission scans, we set excitation at 350 nm and measured emission from 417 to 530 nm with an integration time of 1.0 s nm−1. To determine the effect of ligands, spectra were measured after 15 min incubation with different compounds (at saturating concentrations: [(−)-isoproterenol]=100 μM; [ICI-118,551]=10 μM; [salbutamol]=500 nM). Fluorescence intensity was corrected for background fluorescence from buffer and ligands in all experiments. The curves shown in FIG. 22B are each the average of triplicate experiments performed in parallel. λmax values and intensity changes for β2AR-T4L and β2AR365, each incubated with different ligands, are tabulated in Table 7.

TABLE 7
λmax ± SD forλmax ± SD for
Ligand2AR365 (nm)β2AR-T4L (nm)
none448 ± 2447 ± 2
(−)-isoproteronol453 ± 2455 ± 2
ICI-118,551447 ± 1446 ± 1
salbutamol449 ± 1449 ± 1
Intensity at λmaxLigand/Intensity at λmaxnone
Ligandβ2AR365β2AR-T4L
(−)-isoproteronol0.758 ± 0.0070.824 ± 0.006
ICI-118,5511.013 ± 0.0081.028 ± 0.008
salbutamol0.950 ± 0.0130.928 ± 0.009
Bimane fluorescence responses for unliganded β2AR365 and β2AR-T4L, incubated for 15 min with different ligands.
Top panel shows the λmax for fluorescence emission spectra (excitation at 350 nm and emission from 417 to 530 nm) collected after 15 min incubation with ligand.
Each value is mean ± standard deviation for triplicate experiments performed in parallel.
Bottom panel shows the change in fluorescence intensity after incubation with ligand, represented as the ratio of Intensity at λmax of the ligand to Intensity at λmax of the control no ligand (“none”) response.

Comparing the Proteolytic Stability of Unliganded β2AR and β2AR-T4L.

The limited trypsin proteolysis protocol was adapted from Jiang et al., Biochemistry 44, 1163 (2005). Carazolol-bound β2AR-T4L or wild-type β2AR (each at 30 mg/ml) were diluted 10-fold into HLS buffer (see above) and TPCK-trypsin was added at a 1:1000 ratio (wt:wt). The digests were incubated at room temperature. At various time points, aliquots were removed and flash frozen on dry ice/ethanol. After the last aliquot was removed, all samples were thawed, and an equal volume of 10% SDS/PAGE loading buffer was added to each. Samples were then analyzed by electrophoresis on 12% polyacrylamide gels, followed by staining with Coomassie blue (FIG. 16).

Comparing the Stability of Unliganded β2AR and β2AR-T4L

Unliganded β2AR365 and β2AR-T4L were each purified as described above for the bimane experiments. 200 μl 0.02 mg/ml receptor in HLS buffer was incubated at 37° C. on a heating block. At the time points indicated in FIG. 17, samples were briefly spun and gently vortexed and 16.5 μl was removed and diluted 18.2-fold in HLS (300 μl total). Then 4×5 μl was removed for determination of total binding and 2×5 μl was removed for nonspecific binding. To measure soluble binding, 5 μl diluted receptor was added to 105 μl HLS (400-fold final dilution of receptor) containing 10 nM [3H]DHA±10 μM cold alprenolol. Reactions were incubated 30 min at RT, then on ice until processing. 100 μl of each reaction was applied to a 1 ml G50 column to separate protein from residual unbound [3H]DHA, and receptor was eluted using 1.1 ml ice-cold HLS. Bound [3H]DHA was quantified on a Beckman LS6000 scintillation counter.

Carazolol Dissociation from the “Wild-Type” Receptor β2AR365

β2AR365 was purified with carazolol bound, according to the protocol described above for β2ART4L. Carazolol-bound receptor (at approximately 50 μM concentration) was dialyzed in the dark against 1L dialysis buffer (20 mM HEPES pH7.5, 100 mM NaCl, 0.1% dodecylmaltoside, 300 micromolar alprenolol) at room temperature with stirring. At indicated time points, two samples were removed from the parafilm-sealed open-ended dialysis chamber, diluted into fresh dialysis buffer, and carazolol emission spectra were obtained on a Spex FluoroMax spectrofluorometer (using excitation at 330 nm and emission from 335 to 400 nm). As internal standards for every time point, samples were removed for determination of protein concentration using the Bio-Rad Protein DC kit (FIG. 19).

CAM and UCM Mutants

The CAMs (constitutively active mutants) described in the literature that are the basis for FIG. 26A and the associated discussion are: L124A (Tao, et al., Mol Endocrinol 14, 1272 (2000)), C116F (Zuscik, et al., J Biol Chem 273, 3401 (1998)), D130A (Rasmussen et al., Mol Pharmacol 56, 175 (1999)), L272C (Jensen et al., J Biol Chem 276, 9279 (2001)), and C285T (Shi et al., J Biol Chem 277, 40989 (2002)). The UCMs (uncoupling mutations) from the literature that were used for FIG. 26C are: D79N (Chung, et al., J Biol Chem 263, 4052 (1988)), F139A (Moro, et al., J Biol Chem 269, 6651 (1994)), T1641 (Green, et al., J Biol Chem 268, 23116 (1993)), N318K (Strader et al., Proc Natl Acad Sci U S A 84, 4384 (1987)), N322A (Barak, et al., Biochemistry 34, 15407 (1995)), P323A (Barak, et al., Biochemistry 34, 15407 (1995)), Y326A (Barak, et al., Biochemistry 34, 15407 (1995)), L339A (Gabilondo et al., Proc Natl Acad Sci U S A 94, 12285 (1997)), and L340A (Gabilondo et al., Proc Natl Acad Sci U S A 94, 12285 (1997)).

Biochemical and Structural Analysis of β2AR-T4L

The β2AR fusion protein in which T4 Lysozyme replaces most of the third intracellular loop of the GPCR (“β2AR-T4L”) retains near-native pharmacologic properties. The β2AR-T4L protein was crystallized in lipidic cubic phase, as described in the Examples above, and the resulting 2.4 Å resolution crystal structure reveals the interface between the receptor and the ligand carazolol, a partial inverse agonist. The efficacy of a ligand describes the effect of the ligand on the functional properties of a GPCR. For purposes of the Examples only, agonists are defined as ligands that fully activate the receptor; partial agonists induce submaximal activation even at saturating concentrations; inverse agonists inhibit basal receptor activity, and antagonists have no effect on basal activity, but competitively block access of other ligands. Carazolol, is defined as a partial inverse agonist because it suppresses only 50% of the basal activity of the β2AR. Analysis of mutagenesis data in light of the structure clarifies the roles of different amino acids in inverse agonist binding, and implies that rearrangement of the binding pocket accompanies agonist binding. In addition, the structure reveals how mutations known to cause constitutive activity or uncoupling of agonist binding and G-protein activation are distributed between the ligand-binding pocket and the cytoplasmic surface of the protein, such that changes in side chains due to interaction with the ligand can be transmitted through the structure to the site of G protein interaction.

Cloning of β2AR-T4L

DNA encoding the T4L protein (C54T, C97A) (Matsumura, et al., Proc Natl Acad Sci U S A 86, 6562 (1989)) was initially cloned into the human β2AR gene, guided by comparison of ICL3 length and sequence among class A GPCRs (Horn et al., Nucleic Acids Res 31, 294 (2003)): residues 2345.23-2596.21 of the β2AR were replaced by residues 2-164 of T4L (construct “E3” in FIG. 21A). In addition, the receptor was truncated at position 365, which aligns approximately with the position of the rhodopsin carboxyl terminus. Although these modifications resulted in a receptor that was expressed efficiently in Sf9 cells, further optimization was carried out to reduce the length of the junction between the receptor and the T4L termini, as described in the methods above. Several candidate constructs are illustrated in FIG. 21A, and selected immunofluorescence images of transfected, permeabilized HEK293 cells are shown in FIG. 21B. Relative to the initial construct, we could remove three residues from the cytoplasmic end of helix V, three residues from the C-terminal end of T4L, and three residues from the N terminus of helix VI, all without losing significant cell-surface expression. The final construct used for crystallization trials (“β2AR-T4L”) has residues 2315.70-2626.24 of the β2AR replaced by amino acids 2-161 of T4L (“1D” in FIG. 21A). Similar reduction of flexibility through minimization of linker length has been important in previous crystallization studies on soluble fusion proteins (Smyth, et al., Protein Sci 12, 1313 (2003)).

Functional Properties of β2AR-T4L

We measured saturation binding of [3H]DHA to the β2AR-T4L, as well as competition binding of the inverse agonist ICI-118,551 and several agonists (FIG. 22A, FIG. 15 and Table 6). The results show that β2AR-T4L has wild-type affinity for the antagonist [3H]DHA and the inverse agonist ICI-118,551, whereas the affinity for both agonists (isoproterenol, epinephrine, formoterol) and a partial agonist (salbutamol) is two to three-fold higher relative to wild-type β2AR. Higher agonist binding affinity is a property associated with constitutively active mutants (CAMs) of GPCRs. CAMs of the β2AR also exhibit elevated basal, agonist-independent activation of Gs, and typically have lower expression levels and reduced stability (Gether et al., J Biol Chem 272, 2587 (1997); Rasmussen et al., Mol Pharmacol 56, 175 (1999)). β2ART4L exhibits binding properties of a CAM, but it expresses at levels exceeding 1 mg per liter of Sf9 cell culture, is more resistant to trypsin proteolysis than the wild-type β2AR (FIG. 16), and retains binding activity in detergent at 37° C. as well as the wild-type receptor (FIG. 17).

β2AR-T4L did not couple to Gs, as expected due to the replacement of ICL3 by T4L. To assess whether the fused protein alters receptor function at the level of its ability to undergo conformational changes, we used a covalently attached fluorescent probe as a reporter for ligand-induced structural changes. Fluorophores attached at Cys2656.27, at the cytoplasmic end of helix VI, detect agonist-induced conformational changes that correlate with the efficacy of the agonist towards G protein activation (Ghanouni et al., J Biol Chem 276, 24433 (2001); Ghanouni, et al., Proc Natl Acad Sci U S A 98, 5997 (2001); Swaminath et al., J Biol Chem 279, 686 (2004); Swaminath et al., J Biol Chem 280, 22165 (2005)). Detergent-solubilized β2AR365 (wild-type receptor truncated at 365) and β2AR-T4L were each labeled with monobromobimane. Addition of the agonist isoproterenol to purified β2AR365 induces a decrease in fluorescence intensity and a shift in λmax for the attached bimane probe (FIG. 22B and Table 7). These changes in intensity and λmax are consistent with an agonist-induced increase in polarity around bimane. A smaller change is observed with the partial agonist salbutamol, while the inverse agonist ICI-118,551 had little effect. For the β2AR-T4L, there are subtle differences in the baseline spectrum of the bimane-labeled fusion protein, as might be expected if the environment around Cys2656.27 is altered by T4L. However, the full agonist isoproterenol induces a qualitatively similar decrease in intensity and rightward shift in λmax. Thus the presence of the fused T4L does not prevent agonist-induced conformational changes. The partial agonist salbutamol induced larger responses in β2AR-T4L than were observed in wild-type β2AR, and there was a small increase in fluorescence in response to the inverse agonist 10-118,551. These are properties observed in CAMs (Gether et al., J Biol Chem 272, 2587 (1997); Samama, et al., J Biol Chem 268, 4625 (1993)) and are consistent with the higher affinities for agonists and partial agonists exhibited by β2AR-T4L. Therefore, we conclude that the T4L fusion induces a partial constitutively active phenotype in the β2AR, likely caused by changes at the cytoplasmic ends of helices V and VI.

Comparison Between β2AR-T4L and β2AR-Fab Structures

The β2AR-T4L fusion strategy is validated by comparison of its structure to the structure of wild-type β2AR complexed with a Fab that recognizes a three dimensional epitope consisting of the amino and carboxyl-terminal ends of ICL3, determined at an anisotropic resolution of 3.4 Å/3.7 Å (Rasmussen et al., Nature, 7168:355-6 (2007)). FIG. 23A illustrates the similarity between the fusion and antibody complex approaches to β2AR crystallization, in that both strategies rely on attachment (covalent or non-covalent, respectively) of a soluble protein partner between helices V and VI. A major difference between the two structures is that the extracellular loops and the carazolol ligand could not be modeled in the β2AR-Fab complex, whereas these regions are resolved in the structure of β2AR-T4L. Nonetheless, it is clear that the T4L insertion does not significantly alter the receptor. Superposition of the two structures (FIG. 18) illustrates that the transmembrane helices of the receptor components are very similar (RMSD=0.8 Å for 154 common modeled transmembrane Cα positions, versus 2.3 Å between β2AR-T4L and the 154 equivalent residues in rhodopsin), especially when the modest resolution of the Fab complex is taken into account.

There is one significant difference between the Fab-complex and chimeric receptor structures that can be attributed to the presence of T4L. The cytoplasmic end of helix VI is pulled outward as a result of the fusion to the carboxyl terminus of T4L, which alters the packing of Phe2646.26 at the end of helix VI (FIG. 23B). In the Fab-complex β2AR, interactions between Phe2646.26 and residues in helix V, helix VI, and ICL2 may be important in maintaining the β2AR in the basal state. The loss of these packing interactions in β2AR-T4L could contribute to the higher agonist binding affinity characteristic of a CAM.

An unexpected difference between the structure of rhodopsin and the β2AR-T4L involves the sequence E/DRY found at the cytoplasmic end of helix III in 71% of class A GPCRs. In rhodopsin, Glu1343.49 and Arg1353.50 form a network of hydrogen bond and ionic interactions with Glu2476.36 at the cytoplasmic end of helix VI. These interactions have been referred to as an “ionic lock” that stabilizes the inactive state of rhodopsin and other class A members (Ballesteros et al., J Biol Chem 276, 29171 (2001)). However, the arrangement of the homologous residues is significantly different in β2AR-T4L: Arg13 13.56 interacts primarily with Asp1303.49 and a sulfate ion rather than with Glu2686.36, and the distance between helix III and helix VI is greater than in rhodopsin (FIG. 23C). The fact that similar ionic lock structures were obtained using two different approaches suggests that a broken ionic lock is a genuine feature of the carazolol-bound state of the receptor.

Ligand Binding to the β2AR

The β2AR-T4L fusion protein was purified and crystallized in complex with the inverse agonist carazolol. Carazolol stabilizes the β2AR against extremes of pH and temperature, perhaps related to its unusually high binding affinity (Kd<0.1 nM) and slow dissociation kinetics (t1/2˜30 h) (FIG. 19). The interactions between carazolol and β2AR-T4L are depicted schematically in FIG. 24. The carbazole ring system is oriented roughly perpendicular to the plane of the membrane, and the alkylamine chain (atoms 15-22 in the model) is nearly parallel to the heterocycle (FIG. 25A-B). As described in Example 3, above, carazolol was modeled into the electron density as the (S)-(−) isomer due to the higher affinity of this enantiomer, despite the fact that a racemic mixture of the ligand was used in crystallization. Asp 1133.32, Tyr3167.43, and Asn3127.39 present a constellation of polar functional groups to the alkylamine and alcohol moieties of the ligand, with Asp1133.32 and Asn3127.39 sidechains forming close contacts (<3 Å) with O17 and N19 atoms of carazolol (FIGS. 24 and 25A-B). Asp 1133.32 was one of the first β2AR residues shown to be important for ligand binding; notably the D113N mutation causes complete loss of detectable affinity for antagonists (Strader et al., Proc Natl Acad Sci U S A 84, 4384 (1987)) and a decrease in the potency of agonists towards cell-based G protein activation by over 4 orders of magnitude (Strader et al., J Biol Chem 263, 10267 (1988)). Likewise, mutations of Asn3127.39 perturb β2AR binding to agonists and antagonists: changes to nonpolar amino acids (Ala or Phe) reduce affinities to undetectable levels, while retention of a polar functionality (Thr or Gln) gives partial affinity (Suryanarayana, et al., Mol Pharmacol 44, 111 (1993)). On the opposite end of the ligand near helix V, N7 of the carbazole heterocycle forms a hydrogen bond with the side chain hydroxyl of Ser2035.42. Interestingly, mutations of Ser2035.42 specifically decrease β2AR affinity towards catecholamine agonists and aryloxyalkylamine ligands with nitrogen-containing heterocycles such as pindolol (Liapakis et al., J Biol Chem 275, 37779 (2000)), and by implication carazolol. Thus, the polar interactions between carazolol and the receptor observed in the crystal structure agree with the known biochemical data. The contribution of Tyr3167.43 to antagonist and agonist affinity remains to be tested; this residue is conserved as tyrosine in all sequenced adrenergic receptor genes (Horn et al., Nucleic Acids Res 31, 294 (2003)).

FIG. 25C shows the tight packing between carazolol and surrounding amino acids that buries 790 Å2 of surface area from solvent; specific contacts are depicted schematically in FIG. 24. Notable among the hydrophobic residues contacting carazolol are Val1143.33, Phe2906.52, and Phe1935.32. The side chain of Val1143.33 from helix III makes multiple contacts with the C8-C13 ring of the carbazole heterocycle, and Phe2906.52 from helix VI forms an edge-to-face aromatic interaction with the same ring. As a result, these two amino acids form a hydrophobic “sandwich” with the portion of the aryl moiety that is common to many adrenergic antagonists. Mutation of Val1143.33 to alanine was shown to decrease β2AR affinity towards the antagonist alprenolol by an order of magnitude, as well as lowering affinity for the agonist epinephrine 300-fold (P. Chelikani et al., Proc Natl Acad Sci U S A 104, 7027 (2007)). Phe1935.32 is different from other carazolol contact residues in that it is located on the ECL2, in the path of hormone accessibility to the binding pocket. This amino acid contributes more buried surface area than any other residue to the interface between β2AR-T4L and carazolol (see Table 8). Therefore, Phe1935.32 is likely to contribute significantly to the energy of β2AR-carazolol complex formation, and the position of this residue on the extracellular side of the binding site may allow it to act as a gate that contributes to the unusually slow dissociation of the ligand (FIG. 19).

TABLE 8
β2AR residueSurface area buried (Å2)
Trp1093.2821.4
Thr1103.295.7
Asp1133.3219.3
Val1143.3325.5
Val1173.368.5
Thr1183.371.9
Phe1935.3251.2
Thr1955.347.4
Tyr1995.387.6
Ala2005.3910.0
Ser2035.429.0
Ser2045.434.6
Ser2075.466.3
Trp2866.483.1
Phe2896.5120.0
Phe2906.5219.0
Phe2936.5518.7
Tyr3087.3514.4
Asn3127.3922.5
Tyr3167.436.5
Buried surface area contributions at the β2AR-T4L/carazolol interface.
Solvent accessible surface area calculations were done with the CNS software package (Brunger et al., Acta Crystallogr D Biol Crystallogr 54, 905 (1998)), using a probe radius of 1.4 Å.
Buried surface area contributions of individual residues were determined by calculating solvent-accessible surface area per residue for the full β2ART4L/carazolol model, and subtracting these numbers from the calculated values for the receptor model without carazolol.

Analysis of the binding pocket provides insights into the structural basis for pharmacologic selectivity between the β2AR and closely related adrenergic receptors such as the β1AR. The affinities of these two receptors for certain ligands, such as ICI-118,551, betaxolol and RO363 (Sugimoto et al., J Pharmacol Exp Ther 301, 51 (2002)), differ by up to 100-fold. Curiously, all of the amino acids in the carazolol binding pocket are conserved between the β1AR and β2AR (see FIG. 20). The majority of the 94 amino acid differences between the β1AR and β2AR are found in the cytoplasmic and extracellular loops. While residues that differ in the transmembrane segments generally face the lipid bilayer, eight residues lie at the interface between helices and may influence helix packing. The structural basis for pharmacologic differences between β1AR and β2AR must, therefore, arise from amino acid differences in the entrance to the binding pocket or subtle differences in the packing of helices. Evidence for the latter comes from chimeric receptor studies (Frielle, et al., Proc Nad Acad Sci U S A 85, 9494 (1988)) in which successive exchange of helices between β1AR and β2ARs led to a gradual change in affinity for the β2AR selective ICI-118,551 and the β1AR selective betaxolol.

As discussed above, β2AR-T4L shows CAM-like properties with respect to agonist binding affinities, suggesting that the unliganded β2AR-T4L may exist in a more active conformation than the wild type-β2AR. Nevertheless, as shown in FIG. 22B, β2AR-T4L can be stabilized in an inactive conformation by an inverse agonist. Since β2AR-T4L was crystallized with bound carazolol, a partial inverse agonist, the structure most likely represents an inactive state. This is consistent with the similarity of the β2AR-T4L and β2AR-Fab5 carazolol-bound structures. To assess whether conformational changes are required to accommodate catecholamines, a model of isoproterenol was placed in the binding site such that common atoms (16-22 in FIG. 24) were superimposed onto the analogous carazolol coordinates in the crystal structure (FIG. 25D). Residues Ser2045.43 and Ser2075.46 are critical for catecholamine binding and activation of the β2AR, with Ser2045.43 hydrogen bonding to the meta-hydroxyl and Ser2075.46 to the para-hydroxyl of the catechol ring, respectively (Strader, et al., J Biol Chem 264, 13572 (1989)). In our model, the catechol hydroxyls of isoproterenol face the appropriate serines on helix V, but the distances are too long for hydrogen bonding (6.8 Å from meta-hydroxyl oxygen to the sidechain oxygen of Ser2045.43, 4.8 Å from the para-hydroxyl oxygen to the sidechain oxygen of Ser2075.46). In addition, Asn2936.55 and Tyr3087.35, two residues expected to form selective interactions with agonists based on the literature (Wieland, et al., Proc Nad Acad Sci U S A 93, 9276 (1996); Kikkawa, et al., Mol Pharmacol 53, 128 (1998)), are too distant to form productive polar or hydrophobic contacts with the modeled isoproteronol molecule. These observations suggest that agonist binding requires changes in the binding site relative to the carazolol-bound structure, unless common structural components of agonists and inverse agonists bind in a significantly different manner.

Structural Insights into β2AR Activation

Analysis of mutations that affect β2AR function provides insights into structural rearrangements that are likely to occur during receptor activation. FIG. 26A illustrates the location of amino acids for which mutations lead to elevated basal, agonist-independent activity (constitutively active mutations, CAMs), as well as amino acids for which mutations impair agonist activation (uncoupling mutations, UCMs). Residues for which CAMs have been described are likely to be involved in interactions that maintain the receptor in the inactive conformation. These amino acids are centrally located on helices III and VI. In contrast, positions in which UCMs have been observed are likely to form intramolecular interactions that stabilize the active state. A cluster of UCMs are found at the cytoplasmic end of helix VII. Neither CAMs nor UCMs are directly involved in agonist binding. Although the CAMs and UCMs are not directly connected in sequence, it is evident from the structure that they are linked through packing interactions, such that movements in one will likely affect the packing of others. For example, FIG. 26A (right panel) shows all amino acids with atoms within 4 Å of the two centrally located CAMs, Leu1243.43 (Tao, et al., Mol Endocrinol 14, 1272 (2000)) and Leu2726.34 (Jensen et al., J Biol Chem 276, 9279 (2001)). Several amino acids that pack against these CAMs also interact with one or more UCMs. Trp2866.48 lies at the base of the binding pocket. It has been proposed that agonist binding leads to a change in the rotameric state of Trp2866.48 with subsequent changes in the angle of the helical kink formed by Pro2886.86 (Shi et al., J Biol Chem 277, 40989 (2002)). It is likely that an agonist-induced change in the rotameric state of Trp2866.48 will be linked to changes in sidechains of CAMs and UCMs through packing interactions and propagated to the cytoplasmic ends of the helices and the associated intracellular loops that interact with G proteins and other signaling molecules.

In the structures of both rhodopsin and the β2AR, a cluster of water molecules lies near the most highly conserved class A GPCR residues (FIG. 26B). It has been proposed that these water molecules may play a role in the structural changes involved in receptor activation (Pardo, et al., Chembiochem 8, 19 (2007)). FIG. 26C shows the network of potential hydrogen bonding interactions that link Trp2866.48 with conserved amino acids extending to the cytoplasmic ends of helices. UCMs have been identified for three amino acids linked by this network—N3227.49, P3237.80, and Y3267.83 (Barak et al., Biochemistry 34, 15407 (1995)). This relatively loose-packed, water filled region is likely to be important in allowing conformational transitions, as there will be fewer steric restraints to sidechain repacking.

All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

APPENDIX 1
HEADERMEMBRANE PROTEIN/HYDROLASE 05-OCT-07 2RH1
TITLEHIGH RESOLUTION CRYSTAL STRUCTURE OF HUMAN B2-ADRENERGIC G
TITLE2 PROTEIN-COUPLED RECEPTOR
COMPND  MOL_ID: 1;
COMPND 2 MOLECULE: BETA-2-ADRENERGIC RECEPTOR/T4-LYSOZYME CHIMERA;
COMPND 3 CHAIN: A;
COMPND 4 SYNONYM: BETA-2 ADRENERGIC RECEPTOR, BETA-2 ADRENOCEPTOR,
COMPND 5 BETA-2 ADRENORECEPTOR/LYSIS PROTEIN, MURAMIDASE,
COMPND 6 ENDOLYSIN;
COMPND 7 ENGINEERED: YES;
COMPND 8 MUTATION: YES
SOURCE MOL_ID: 1;
SOURCE 2 ORGANISM_SCIENTIFIC: HOMO SAPIENS, ENTEROBACTERIA PHAGE T4;
SOURCE 3 ORGANISM_COMMON: HUMAN,;
SOURCE 4 STRAIN: ,;
SOURCE 5 GENE: ADRB2, ADRB2R, B2AR/E;
SOURCE 6 EXPRESSION_SYSTEM: SPODOPTERA FRUGIPERDA;
SOURCE 7 EXPRESSION_SYSTEM_COMMON: FALL ARMYWORM;
SOURCE 8 EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID;
SOURCE 9 EXPRESSION_SYSTEM_PLASMID: PFASTBAC1;
SOURCE10 OTHER_DETAILS: THE CONSTRUCT HAS BEEN OBTAINED BY
SOURCE11 OVERLAPPING EXTENSION PCR
KEYWDS GPCR, 7TM, ADRENERGIC, FUSION, LIPIDIC CUBIC PHASE, LIPIDIC,
KEYWDS2 MESOPHASE, CHOLESTEROL, MEMBRANE PROTEIN, MEMBRANE PROTEIN
KEYWDS3/HYDROLASE COMPLEX
EXPDTAX-RAY DIFFRACTION
AUTHOR V. CHEREZOV, D. M. ROSENBAUM, M. A. HANSON, S. G. F. RASMUSSEN,
AUTHOR2 F. S. THIAN, T. S. KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS, B. K. KOBILKA,
AUTHOR3 R. C. STEVENS
REVDAT5 07-OCT-08 2RH11REMARK
REVDAT4 11-DEC-07 2RH11REMARK
REVDAT3 04-DEC-07 2RH11JRNL
REVDAT2 06-NOV-07 2RH11JRNL HELIX SHEET
REVDAT1 30-OCT-07 2RH10
JRNLAUTH V. CHEREZOV, D. M. ROSENBAUM, M. A. HANSON, S. G. RASMUSSEN,
JRNLAUTH 2 F. S. THIAN, T. S. KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS,
JRNLAUTH 3 B. K. KOBILKA, R. C. STEVENS
JRNLTITL HIGH-RESOLUTION CRYSTAL STRUCTURE OF AN ENGINEERED
JRNLTITL 2 HUMAN BETA2-ADRENERGIC G PROTEIN-COUPLED RECEPTOR.
JRNLREF SCIENCE V. 318 1258 2007
JRNLREFN ASTM SCIEAS US ISSN 0036-8075
REMARK1
REMARK1REFERENCE 1
REMARK1 AUTH D. M. ROSENBAUM, V. CHEREZOV, M. A. HANSON,
REMARK1 AUTH 2 S. G. F. RASMUSSEN, F. S. THIAN, T. S. KOBILKA, H. J. CHOI,
REMARK1 AUTH 3 X. J. YAO, W. I. WEIS, R. C. STEVENS, B. K. KOBILKA
REMARK1 TITL GPCR ENGINEERING YIELDS HIGH-RESOLUTION STRUCTURAL
REMARK1 TITL 2 INSIGHTS INTO BETA2 ADRENERGIC RECEPTOR FUNCTION.
REMARK1 REF TO BE PUBLISHED
REMARK1 REFN
REMARK2
REMARK2RESOLUTION. 2.40 ANGSTROMS.
REMARK3
REMARK3REFINEMENT.
REMARK3 PROGRAM: REFMAC 5.2.0019
REMARK3 AUTHORS: MURSHUDOV, VAGIN, DODSON
REMARK3
REMARK3 REFINEMENT TARGET: MAXIMUM LIKELIHOOD
REMARK3
REMARK3DATA USED IN REFINEMENT.
REMARK3 RESOLUTION RANGE HIGH (ANGSTROMS): 2.40
REMARK3 RESOLUTION RANGE LOW (ANGSTROMS): 19.95
REMARK3 DATA CUTOFF (SIGMA(F)): 0.000
REMARK3 COMPLETENESS FOR RANGE (%): 99.8
REMARK3 NUMBER OF REFLECTIONS: 26506
REMARK3
REMARK3FIT TO DATA USED IN REFINEMENT.
REMARK3 CROSS-VALIDATION METHOD: THROUGHOUT
REMARK3 FREE R VALUE TEST SET SELECTION: RANDOM
REMARK3 R VALUE (WORKING + TEST SET): 0.198
REMARK3 R VALUE (WORKING SET): 0.196
REMARK3 FREE R VALUE: 0.232
REMARK3 FREE R VALUE TEST SET SIZE (%): 4.900
REMARK3 FREE R VALUE TEST SET COUNT: 1310
REMARK3
REMARK3FIT IN THE HIGHEST RESOLUTION BIN.
REMARK3 TOTAL NUMBER OF BINS USED:20
REMARK3 BIN RESOLUTION RANGE HIGH:2.40
REMARK3 BIN RESOLUTION RANGE LOW:2.46
REMARK3 REFLECTION IN BIN (WORKING SET): 1829
REMARK3 BIN COMPLETENESS (WORKING + TEST) (%): 98.76
REMARK3 BIN R VALUE (WORKING SET): 0.2700
REMARK3 BIN FREE R VALUE SET COUNT: 78
REMARK3 BIN FREE R VALUE: 0.3010
REMARK3
REMARK3NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT.
REMARK3 ALL ATOMS: 3804
REMARK3
REMARK3B VALUES.
REMARK3 FROM WILSON PLOT (A**2): 63.91
REMARK3 MEAN B VALUE (OVERALL, A**2): 63.91
REMARK3 OVERALL ANISOTROPIC B VALUE.
REMARK3 B11 (A**2): 0.43000
REMARK3 B22 (A**2): −3.46000
REMARK3 B33 (A**2): 4.25000
REMARK3 B12 (A**2): 0.00000
REMARK3 B13 (A**2): 2.26000
REMARK3 B23 (A**2): 0.00000
REMARK3
REMARK3ESTIMATED OVERALL COORDINATE ERROR.
REMARK3 ESU BASED ON R VALUE (A): 0.295
REMARK3 ESU BASED ON FREE R VALUE (A):0.220
REMARK3 ESU BASED ON MAXIMUM LIKELIHOOD (A): 0.203
REMARK3 ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 18.501
REMARK3
REMARK3CORRELATION COEFFICIENTS.
REMARK3 CORRELATION COEFFICIENT FO-FC:  0.963
REMARK3 CORRELATION COEFFICIENT FO-FC FREE: 0.944
REMARK3
REMARK3RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT
REMARK3 BOND LENGTHS REFINED ATOMS (A): 3843; 0.013; 0.022
REMARK3 BOND LENGTHS OTHERS (A): 2622; 0.000; 0.020
REMARK3 BOND ANGLES REFINED ATOMS (DEGREES): 5219; 1.500; 2.000
REMARK3 BOND ANGLES OTHERS (DEGREES): 6377; 4.099; 3.002
REMARK3 TORSION ANGLES, PERIOD 1 (DEGREES): 441; 3.876; 5.000
REMARK3 TORSION ANGLES, PERIOD 2 (DEGREES): 154; 31.604; 23.182
REMARK3 TORSION ANGLES, PERIOD 3 (DEGREES): 627; 11.383; 15.000
REMARK3 TORSION ANGLES, PERIOD 4 (DEGREES):  22; 12.164; 15.000
REMARK3 CHIRAL-CENTER RESTRAINTS (A**3): 610; 0.060; 0.200
REMARK3 GENERAL PLANES REFINED ATOMS  (A): 4008; 0.001; 0.020
REMARK3 GENERAL PLANES OTHERS (A): 804; 0.001; 0.020
REMARK3 NON-BONDED CONTACTS REFINED ATOMS (A): 926; 0.187; 0.300
REMARK3 NON-BONDED CONTACTS OTHERS (A): 2443; 0.194; 0.300
REMARK3 NON-BONDED TORSION REFINED ATOMS (A): 1935; 0.180; 0.500
REMARK3 NON-BONDED TORSION OTHERS (A): 1580; 0.107; 0.500
REMARK3 H-BOND (X . . . Y) REFINED ATOMS (A): 194; 0.164; 0.500
REMARK3 H-BOND (X . . . Y) OTHERS (A): NULL; NULL; NULL
REMARK3 POTENTIAL METAL-ION REFINED ATOMS (A): NULL; NULL; NULL
REMARK3 POTENTIAL METAL-ION OTHERS (A): NULL; NULL; NULL
REMARK3 SYMMETRY VDW REFINED ATOMS (A): 7; 0.155; 0.300
REMARK3 SYMMETRY VDW OTHERS (A): 29; 0.140; 0.300
REMARK3 SYMMETRY H-BOND REFINED ATOMS (A): 6; 0.192; 0.500
REMARK3 SYMMETRY H-BOND OTHERS (A): NULL; NULL; NULL
REMARK3 SYMMETRY METAL-ION REFINED ATOMS (A): NULL; NULL; NULL
REMARK3 SYMMETRY METAL-ION OTHERS (A): NULL; NULL; NULL
REMARK3
REMARK3ISOTROPIC THERMAL FACTOR RESTRAINTS. COUNT RMS WEIGHT
REMARK3 MAIN-CHAIN BOND REFINED ATOMS (A**2): 2884; 2.352; 2.000
REMARK3 MAIN-CHAIN BOND OTHER ATOMS (A**2): 896; 0.097; 2.000
REMARK3 MAIN-CHAIN ANGLE REFINED ATOMS (A**2): 3571; 2.767; 2.500
REMARK3 SIDE-CHAIN BOND REFINED ATOMS (A**2): 1974; 4.582; 2.000
REMARK3 SIDE-CHAIN ANGLE REFINED ATOMS (A**2): 1648; 5.849; 2.500
REMARK3
REMARK3ANISOTROPIC THERMAL FACTOR RESTRAINTS. COUNT RMS WEIGHT
REMARK3 RIGID-BOND RESTRAINTS (A**2): NULL; NULL; NULL
REMARK3 SPHERICITY; FREE ATOMS (A**2): NULL; NULL; NULL
REMARK3 SPHERICITY; BONDED ATOMS  (A**2): NULL; NULL; NULL
REMARK3
REMARK3NCS RESTRAINTS STATISTICS
REMARK3 NUMBER OF DIFFERENT NCS GROUPS: NULL
REMARK3
REMARK3TLS DETAILS
REMARK3 NUMBER OF TLS GROUPS: 3
REMARK3
REMARK3 TLS GROUP: 1
REMARK3 NUMBER OF COMPONENTS GROUP: 2
REMARK3 COMPONENTS C SSSEQI TO C SSSEQI
REMARK3 RESIDUE RANGE: A 1002 A 1011
REMARK3 RESIDUE RANGE: A 1062 A 1161
REMARK3 ORIGIN FOR THE GROUP (A): −23.6840 58.4050 30.4850
REMARK3 T TENSOR
REMARK3 T11: −0.2208 T22: −0.2598
REMARK3 T33: −0.7033 T12:  0.0432
REMARK3 T13: −0.0594 T23: −0.0241
REMARK3 L TENSOR
REMARK3 L11:  3.1220 L22:  2.6914
REMARK3 L33:  5.9787 L12:  0.8643
REMARK3 L13:  0.6146 L23: −0.8632
REMARK3 S TENSOR
REMARK3 S11: −0.1453 S12: −0.2225 S13:  0.2116
REMARK3 S21:  0.0984 S22: −0.0656 S23: −0.0133
REMARK3 S31: −0.1849 S32: −0.1761 S33:  0.2109
REMARK3
REMARK3TLS GROUP: 2
REMARK3 NUMBER OF COMPONENTS GROUP: 1
REMARK3 COMPONENTS C SSSEQI TO C SSSEQI
REMARK3 RESIDUE RANGE: A 1012 A 1061
REMARK3 ORIGIN FOR THE GROUP (A): −35.0580 69.0010 11.9610
REMARK3 T TENSOR
REMARK3 T11:  0.0414 T22: −0.0871
REMARK3 T33: −0.4908 T12:  0.0577
REMARK3 T13: −0.1559 T23: −0.0085
REMARK3 L TENSOR
REMARK3 L11:  9.6406 L22: 16.6501
REMARK3 L33:  7.1133 L12: −6.5961
REMARK3 L13: −0.9803 L23:  3.2882
REMARK3 S TENSOR
REMARK3 S11: −0.1628 S12: −0.1008 S13: 0.4114
REMARK3 S21: −0.7585 S22: −0.1058 S23: 0.7355
REMARK3 S31: −0.6590 S32: −0.6073 S33: 0.2686
REMARK3
REMARK3 TLS GROUP: 3
REMARK3 NUMBER OF COMPONENTS GROUP: 2
REMARK3 COMPONENTS C SSSEQI TO C SSSEQI
REMARK3 RESIDUE RANGE: A  29 A 230
REMARK3 RESIDUE RANGE: A 263 A 342
REMARK3 ORIGIN FOR THE GROUP (A): −33.0740 20.0130 7.1220
REMARK3 T TENSOR
REMARK3 T11: −0.0103 T22: −0.2341
REMARK3 T33: −0.5401 T12: −0.0025
REMARK3 T13: −0.0974 T23: −0.0034
REMARK3 L TENSOR
REMARK3 L11:  2.3670 L22: 6.1551
REMARK3 L33:  1.9314 L12: 2.1068
REMARK3 L13:  0.8591 L23: 0.7864
REMARK3 S TENSOR
REMARK3 S11: −0.0346 S12:  0.0267 S13: −0.2068
REMARK3 S21: −0.5009 S22:  0.0712 S23:  0.2388
REMARK3 S31:  0.3208 S32:  0.0002 S33: −0.0366
REMARK3
REMARK3BULK SOLVENT MODELLING.
REMARK3 METHOD USED: MASK
REMARK3 PARAMETERS FOR MASK CALCULATION
REMARK3 VDW PROBE RADIUS: 1.40
REMARK3 ION PROBE RADIUS: 0.80
REMARK3 SHRINKAGE RADIUS: 0.80
REMARK3
REMARK3OTHER REFINEMENT REMARKS: HYDROGENS HAVE BEEN ADDED IN THE
REMARK3RIDING POSITIONS. WATER #548 HAS STRONG DIFFERENCE DENSITY BUT
REMARK3WEAK 2FO-FC DENSITY.
REMARK4
REMARK42RH1 COMPLIES WITH FORMAT V. 3.1, 01-AUG-2007
REMARK100
REMARK100THIS ENTRY HAS BEEN PROCESSED BY RCSB.
REMARK100THE RCSB ID CODE IS RCSB044849.
REMARK200
REMARK200EXPERIMENTAL DETAILS
REMARK200 EXPERIMENT TYPE: X-RAY DIFFRACTION
REMARK200 DATE OF DATA COLLECTION:  22-JUN-2007; 18-JUL-2007
REMARK200 TEMPERATURE (KELVIN): 78; 78
REMARK200 PH: 6.75
REMARK200 NUMBER OF CRYSTALS USED:  27
REMARK200
REMARK200 SYNCHROTRON  (Y/N): Y; Y
REMARK200 RADIATION SOURCE: APS; APS
REMARK200 BEAMLINE: 23-ID-B; 23-ID-B
REMARK200 X-RAY GENERATOR MODEL: NULL
REMARK200 MONOCHROMATIC OR LAUE (M/L): M
REMARK200 WAVELENGTH OR RANGE (A):1.03321; 1.03321
REMARK200MONOCHROMATOR: DOUBLE CRYSTAL
REMARK200OPTICS: MIRRORS; MIRRORS
REMARK200
REMARK200DETECTOR TYPE: CCD; CCD
REMARK200DETECTOR MANUFACTURER: MARMOSAIC 300 MM CCD;
REMARK200 MARMOSAIC 300 MM CCD
REMARK200INTENSITY-INTEGRATION SOFTWARE: XDS
REMARK200DATA SCALING SOFTWARE: XDS
REMARK200
REMARK200NUMBER OF UNIQUE REFLECTIONS: 26506
REMARK200RESOLUTION RANGE HIGH(A): 2.400
REMARK200RESOLUTION RANGE LOW(A): 20.000
REMARK200REJECTION CRITERIA (SIGMA(I)): −3.000
REMARK200
REMARK200OVERALL.
REMARK200 COMPLETENESS FOR RANGE (%): 99.2
REMARK200 DATA REDUNDANCY: NULL
REMARK200 R MERGE (I): 0.12700
REMARK200 R SYM (I): NULL
REMARK200 <I/SIGMA(I)> FOR THE DATA SET: 9.6200
REMARK200
REMARK200IN THE HIGHEST RESOLUTION SHELL.
REMARK200 HIGHEST RESOLUTION SHELL, RANGE HIGH (A): 2.40
REMARK200 HIGHEST RESOLUTION SHELL, RANGE LOW (A): 2.50
REMARK200 COMPLETENESS FOR SHELL (%): 99.1
REMARK200 DATA REDUNDANCY IN SHELL: NULL
REMARK200 R MERGE FOR SHELL (I): 0.67800
REMARK200 R SYM FOR SHELL (I): NULL
REMARK200 <I/SIGMA(I)> FOR SHELL:  2.200
REMARK200
REMARK200DIFFRACTION PROTOCOL: SINGLE WAVELENGTH
REMARK200METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR REPLACEMENT
REMARK200SOFTWARE USED: PHASER
REMARK200STARTING MODEL: PDB ENTRIES 1U19, 2LZM
REMARK200
REMARK200REMARK: THIS STRUCTURE IS A PART OF THE ROADMAP/PSI COMMUNITY
REMARK200 OUTREACH PROGRAM, NOT A SPECIFIC PSI TARGET.
REMARK280
REMARK280CRYSTAL
REMARK280SOLVENT CONTENT, VS (%): 59.98
REMARK280MATTHEWS COEFFICIENT, VM (ANGSTROMS**3/DA): 3.07
REMARK280
REMARK280CRYSTALLIZATION CONDITIONS: 30-35% V/V PEG 400, 0.1-0.2 M
REMARK280 NA2SO4, 0.1 M BIS-TRIS PROPANE PH 6.5-7.0, 5-7% 1,4-
REMARK280 BUTANEDIOL, 8-10% CHOLESTEROL, 52-50% MONOOLEIN, PH 6.75,
REMARK280 LIPIDIC MESOPHASE, TEMPERATURE 293 K
REMARK290
REMARK290CRYSTALLOGRAPHIC SYMMETRY
REMARK290SYMMETRY OPERATORS FOR SPACE GROUP: C 1 2 1
REMARK290
REMARK290  SYMOP SYMMETRY
REMARK290 NNNMMM OPERATOR
REMARK290 1555 X, Y, Z
REMARK290 2555 −X, Y, −Z
REMARK290 3555 ½ + X, ½ + Y, Z
REMARK290 4555 ½ − X, ½ + Y, −Z
REMARK290
REMARK290 WHERE NNN -> OPERATOR NUMBER
REMARK290 MMM -> TRANSLATION VECTOR
REMARK290CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS
REMARK290THE FOLLOWING TRANSFORMATIONS OPERATE ON THE ATOM/HETATM
REMARK290RECORDS IN THIS ENTRY TO PRODUCE CRYSTALLOGRAPHICALLY
REMARK290RELATED MOLECULES.
REMARK290 SMTRY111.0000000.000000 0.000000 0.00000
REMARK290 SMTRY210.0000001.000000 0.000000 0.00000
REMARK290 SMTRY310.0000000.000000 1.000000 0.00000
REMARK290 SMTRY12−1.0000000.000000 0.000000 0.00000
REMARK290 SMTRY220.0000001.000000 0.000000 0.00000
REMARK290 SMTRY320.0000000.000000−1.000000 0.00000
REMARK290 SMTRY131.0000000.000000 0.00000053.15900
REMARK290 SMTRY230.0000001.000000 0.00000084.62000
REMARK290 SMTRY330.0000000.000000 1.000000 0.00000
REMARK290 SMTRY14−1.0000000.000000 0.00000053.15900
REMARK290 SMTRY240.0000001.000000 0.00000084.62000
REMARK290 SMTRY340.0000000.000000−1.000000 0.00000
REMARK290
REMARK290REMARK: NULL
REMARK300
REMARK300BIOMOLECULE: 1
REMARK300SEE REMARK 350 FOR THE AUTHOR PROVIDED AND/OR PROGRAM
REMARK300GENERATED ASSEMBLY INFORMATION FOR THE STRUCTURE IN
REMARK300THIS ENTRY. THE REMARK MAY ALSO PROVIDE INFORMATION ON
REMARK300BURIED SURFACE AREA.
REMARK300
REMARK300REMARK: AUTHORS STATE THAT THE BIOLOGICAL UNIT IS UNKNOWN
REMARK350
REMARK350COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWN
REMARK350BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE OF THE
REMARK350MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS
REMARK350GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND
REMARK350CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN.
REMARK350
REMARK350BIOMOLECULE: 1
REMARK350SOFTWARE DETERMINED QUATERNARY STRUCTURE: MONOMERIC
REMARK350SOFTWARE USED: PISA
REMARK350APPLY THE FOLLOWING TO CHAINS: A
REMARK350 BIOMT111.0000000.0000000.0000000.00000
REMARK350 BIOMT210.0000001.0000000.0000000.00000
REMARK350 BIOMT310.0000000.0000001.0000000.00000
REMARK375
REMARK375SPECIAL POSITION
REMARK375THE FOLLOWING ATOMS ARE FOUND TO BE WITHIN 0.15 ANGSTROMS
REMARK375OF A SYMMETRY RELATED ATOM AND ARE ASSUMED TO BE ON SPECIAL
REMARK375POSITIONS.
REMARK375
REMARK375ATOM RES CSSEQI
REMARK375 HOH A 520 LIES ON A SPECIAL POSITION.
REMARK465
REMARK465MISSING RESIDUES
REMARK465THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
REMARK465EXPERIMENT. (M = MODEL NUMBER; RES = RESIDUE NAME; C = CHAIN
REMARK465IDENTIFIER; SSEQ = SEQUENCE NUMBER; I = INSERTION CODE.)
REMARK465
REMARK465 M RES C SSEQI
REMARK465 ASP A −6
REMARK465 TYR A −5
REMARK465 LYS A −4
REMARK465 ASP A −3
REMARK465 ASP A −2
REMARK465 ASP A −1
REMARK465 ALA A 0
REMARK465 MET A 1
REMARK465 GLY A 2
REMARK465 GLN A 3
REMARK465 PRO A 4
REMARK465 GLY A 5
REMARK465 ASN A 6
REMARK465 GLY A 7
REMARK465 SER A 8
REMARK465 ALA A 9
REMARK465 PHE A 10
REMARK465 LEU A 11
REMARK465 LEU A 12
REMARK465 ALA A 13
REMARK465 PRO A 14
REMARK465 ASN A 15
REMARK465 ARG A 16
REMARK465 SER A 17
REMARK465 HIS A 18
REMARK465 ALA A 19
REMARK465 PRO A 20
REMARK465 ASP A 21
REMARK465 HIS A 22
REMARK465 ASP A 23
REMARK465 VAL A 24
REMARK465 THR A 25
REMARK465 GLN A 26
REMARK465 GLN A 27
REMARK465 ARG A 28
REMARK465 ARG A 343
REMARK465 ARG A 344
REMARK465 SER A 345
REMARK465 SER A 346
REMARK465 LEU A 347
REMARK465 LYS A 348
REMARK465 ALA A 349
REMARK465 TYR A 350
REMARK465 GLY A 351
REMARK465 ASN A 352
REMARK465 GLY A 353
REMARK465 TYR A 354
REMARK465 SER A 355
REMARK465 SER A 356
REMARK465 ASN A 357
REMARK465 GLY A 358
REMARK465 ASN A 359
REMARK465 THR A 360
REMARK465 GLY A 361
REMARK465 GLU A 362
REMARK465 GLN A 363
REMARK465 SER A 364
REMARK465 GLY A 365
REMARK470
REMARK470MISSING ATOM
REMARK470THE FOLLOWING RESIDUES HAVE MISSING ATOMS(M = MODEL NUMBER;
REMARK470RES = RESIDUE NAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER;
REMARK470I = INSERTION CODE):
REMARK470 M RES CSSEQI ATOMS
REMARK470 ASP A 29 CG OD1 OD2
REMARK500
REMARK500GEOMETRY AND STEREOCHEMISTRY
REMARK500SUBTOPIC: CLOSE CONTACTS IN SAME ASYMMETRIC UNIT
REMARK500
REMARK500THE FOLLOWING ATOMS ARE IN CLOSE CONTACT.
REMARK500
REMARK500ATM1 RES C SSEQI ATM2 RES C SSEQI
REMARK500 SG1 CYS A  341 O2 PLM A 415 1.88
REMARK500
REMARK500REMARK: NULL
REMARK500
REMARK500GEOMETRY AND STEREOCHEMISTRY
REMARK500SUBTOPIC: TORSION ANGLES
REMARK500
REMARK500TORSION ANGLES OUTSIDE THE EXPECTED RAMACHANDRAN REGIONS:
REMARK500(M = MODEL NUMBER; RES = RESIDUE NAME; C = CHAIN IDENTIFIER;
REMARK500SSEQ = SEQUENCE NUMBER; I = INSERTION CODE).
REMARK500
REMARK500STANDARD TABLE:
REMARK500FORMAT: (10X, I3, 1X, A3, 1X, A1, I4, A1, 4X, F7.2, 3X, F7.2)
REMARK500
REMARK500EXPECTED VALUES: GJ KLEYWEGT AND TA JONES (1996). PHI/PSI-
REMARK500CHOLOGY: RAMACHANDRAN REVISITED. STRUCTURE 4, 1395-1400
REMARK500
REMARK500M RES CSSEQI PSI PHI
REMARK500 VAL A 86 −64.27 −100.05
REMARK500 TYR A 141 −22.19 73.12
REMARK500
REMARK500REMARK: NULL
REMARK600
REMARK600HETEROGEN
REMARK600THE PALMITIC ACID (PLM) AND ACETAMIDE (ACM) GROUPS ARE
REMARK600COVALENTLY LINKED TO THEIR RESPECTIVE CYSTEINE RESIDUES.
REMARK610
REMARK610MISSING HETEROATOM
REMARK610THE FOLLOWING RESIDUES HAVE MISSING ATOMS (M = MODEL NUMBER;
REMARK610RES = RESIDUE NAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER;
REMARK610I = INSERTION CODE):
REMARK610 M RES C SSEQI
REMARK610 12P A 416
REMARK800
REMARK800SITE
REMARK800SITE_IDENTIFIER: AC1
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE 12P A 416
REMARK800SITE_IDENTIFIER: AC2
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE ACM A 411
REMARK800SITE_IDENTIFIER: AC3
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE BU1 A 409
REMARK800SITE_IDENTIFIER: AC5
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE CAU A 408
REMARK800SITE_IDENTIFIER: AC6
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE CLR A 412
REMARK800SITE_IDENTIFIER: AC7
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE CLR A 413
REMARK800SITE_IDENTIFIER: AC9
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE MAL A 401
REMARK800SITE_IDENTIFIER: BC1
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE PLM A 415
REMARK800SITE_IDENTIFIER: BC2
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 402
REMARK800SITE_IDENTIFIER: BC3
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 403
REMARK800SITE_IDENTIFIER: BC4
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 404
REMARK800SITE_IDENTIFIER: BC5
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 405
REMARK800SITE_IDENTIFIER: BC6
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 406
REMARK800SITE_IDENTIFIER: BC7
REMARK800SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 407
REMARK999
REMARK999SEQUENCE THE STRUCTURE IS AN INTERNAL FUSION PROTEIN WITH
REMARK999LYSOZYME. AN OFFSET 1000 HAS BEEN ADDED TO ORIGINAL
REMARK999SEQUENCE DATABASE RESIDUE NUMBERS (2-161) OF THE LYSOZYME
REMARK999PART IN COORDINATES TO DISTINGUISH THE LYSOZYME PART IN THE
REMARK999CHAIN. THEREFORE THE RESIDUES OF LYSOZYME PART HAVE NUMBERS
REMARK999A1002-A1161.
DBREF2RH1A 1 230 UNP P07550 ADRB2_HUMAN  1 230
DBREF2RH1A1002 1161 UNP  P00720 LYS_BPT4 2  161
DBREF2RH1A 263 365 UNP  P07550 ADRB2_HUMAN 263  365
SEQADV2RH1ASPA −6UNPP07550EXPRESSION TAG
SEQADV2RH1TYRA −5UNPP07550EXPRESSION TAG
SEQADV2RH1LYSA −4UNPP07550EXPRESSION TAG
SEQADV2RH1ASPA −3UNPP07550EXPRESSION TAG
SEQADV2RH1ASPA −2UNPP07550EXPRESSION TAG
SEQADV2RH1ASPA −1UNPP07550EXPRESSION TAG
SEQADV2RH1ALAA 0UNPP07550EXPRESSION TAG
SEQADV2RH1GLUA 187UNPP07550ASN187ENGINEERED
SEQADV2RH1THRA 1054UNPP00720CYS54ENGINEERED
SEQADV2RH1ALAA1097UNPP00720CYS97ENGINEERED
SEQRES1A500ASPTYRLYSASPASPASPALAMETGLYGLNPROGLYASN
SEQRES2A500GLYSERALAPHELEULEUALAPROASNARGSERHISALA
SEQRES3A500PROASPHISASPVALTHRGLNGLNARGASPGLUVALTRP
SEQRES4A500VALVALGLYMETGLYILEVALMETSERLEUILEVALLEU
SEQRES5A500ALAILEVALPHEGLYASNVALLEUVALILETHRALAILE
SEQRES6A500ALALYSPHEGLUARGLEUGLNTHRVALTHRASNTYRPHE
SEQRES7A500ILETHRSERLEUALACYSALAASPLEUVALMETGLYLEU
SEQRES8A500ALAVALVALPROPHEGLYALAALAHISILELEUMETLYS
SEQRES9A500METTRPTHRPHEGLYASNPHETRPCYSGLUPHETRPTHR
SEQRES10A500SERILEASPVALLEUCYSVALTHRALASERILEGLUTHR
SEQRES11A500LEUCYSVALILEALAVALASPARGTYRPHEALAILETHR
SEQRES12A500SERPROPHELYSTYRGLNSERLEULEUTHRLYSASNLYS
SEQRES13A500ALAARGVALILEILELEUMETVALTRPILEVALSERGLY
SEQRES14A500LEUTHRSERPHELEUPROILEGLNMETHISTRPTYRARG
SEQRES15A500ALATHRHISGLNGLUALAILEASNCYSTYRALAGLUGLU
SEQRES16A500THRCYSCYSASPPHEPHETHRASNGLNALATYRALAILE
SEQRES17A500ALASERSERILEVALSERPHETYRVALPROLEUVALILE
SEQRES18A500METVALPHEVALTYRSERARGVALPHEGLNGLUALALYS
SEQRES19A500ARGGLNLEUASNILEPHEGLUMETLEUARGILEASPGLU
SEQRES20A500GLYLEUARGLEULYSILETYRLYSASPTHRGLUGLYTYR
SEQRES21A500TYRTHRILEGLYILEGLYHISLEULEUTHRLYSSERPRO
SEQRES22A500SERLEUASNALAALALYSSERGLULEUASPLYSALAILE
SEQRES23A500GLYARGASNTHRASNGLYVALILETHRLYSASPGLUALA
SEQRES24A500GLULYSLEUPHEASNGLNASPVALASPALAALAVALARG
SEQRES25A500GLYILELEUARGASNALALYSLEULYSPROVALTYRASP
SEQRES26A500SERLEUASPALAVALARGARGALAALALEUILEASNMET
SEQRES27A500VALPHEGLNMETGLYGLUTHRGLYVALALAGLYPHETHR
SEQRES28A500ASNSERLEUARGMETLEUGLNGLNLYSARGTRPASPGLU
SEQRES29A500ALAALAVALASNLEUALALYSSERARGTRPTYRASNGLN
SEQRES30A500THRPROASNARGALALYSARGVALILETHRTHRPHEARG
SEQRES31A500THRGLYTHRTRPASPALATYRLYSPHECYSLEULYSGLU
SEQRES32A500HISLYSALALEULYSTHRLEUGLYILEILEMETGLYTHR
SEQRES33A500PHETHRLEUCYSTRPLEUPROPHEPHEILEVALASNILE
SEQRES34A500VALHISVALILEGLNASPASNLEUILEARGLYSGLUVAL
SEQRES35A500TYRILELEULEUASNTRPILEGLYTYRVALASNSERGLY
SEQRES36A500PHEASNPROLEUILETYRCYSARGSERPROASPPHEARG
SEQRES37A500ILEALAPHEGLNGLULEULEUCYSLEUARGARGSERSER
SEQRES38A500LEULYSALATYRGLYASNGLYTYRSERSERASNGLYASN
SEQRES39A500THRGLYGLUGLNSERGLY
HETMAL A 401 23
HETSO4 A 402 5
HETSO4 A 403 5
HETSO4 A 404 5
HETSO4 A 405 5
HETSO4 A 406 5
HETSO4 A 407 5
HETCAU A 408 22
HETBU1 A 409 6
HETBU1 A 410 6
HETACM A 411 4
HETCLR A 412 28
HETCLR A 413 28
HETCLR A 414 28
HETPLM A 415 17
HET12P A 416 21
HETNAM  MAL MALTOSE
HETNAM  SO4 SULFATE ION
HETNAM  CAU (2S)-1-(9H-CARBAZOL-4-YLOXY)-3-(ISOPROPYLAMINO)PROPAN-
HETNAM2 CAU 2-OL
HETNAM  BU1 1,4-BUTANEDIOL
HETNAM  ACM ACETAMIDE
HETNAM  CLR CHOLESTEROL
HETNAM  PLM PALMITIC ACID
HETNAM  12P DODECAETHYLENE GLYCOL
HETSYN  CAU (S)-CARAZOLOL
HETSYN  12P POLYETHYLENE GLYCOL PEG400
FORMUL 2 MAL C12 H22 O11
FORMUL 3 SO4 6(O4 S 2−)
FORMUL 9 CAU C18 H22 N2 O2
FORMUL10 BU1 2(C4 H10 O2)
FORMUL12 ACM C2 H5 N O
FORMUL13 CLR 3(C27 H46 O)
FORMUL16 PLM C16 H32 O2
FORMUL17 12P C24 H50 O13
FORMUL18 HOH *48(H2 O)
HELIX 11 ASP A  29 LYS A  60  132
HELIX 22 VAL A  67 MET A  96  130
HELIX 33 ASN A  103 THR A  136  1  34
HELIX 44 LYS A  147 MET A  171  1 25
HELIX 55 HIS A  178 GLU A  187  1 10
HELIX 66 GLN A  197 GLN A  229  1  33
HELIX 77 LYS A  267 ILE A  298  132
HELIX 88 LYS A  305 ARG A  328  1  24
HELIX 99 PRO A  330 CYS A  341  1  12
HELIX1010 ILE A  1003 GLU A  1011  1  9
HELIX1111 LEU A  1039 ILE A  1050  1  12
HELIX1212 LYS A  1060 ARG A  1080  1   21
HELIX1313 ALA A  1082 SER A  1090  1    9
HELIX1414 ALA A  1093 MET A  1106  1    14
HELIX1515 GLU A  1108 GLY A  1113  1    6
HELIX1616 THR A  1115 GLN A  1123  1    9
HELIX1717 TRP A  1126 ALA A  1134  1    9
HELIX1818 ARG A  1137 GLN A  1141  1    5
HELIX1919 PRO A  1143 THR A  1155  1    13
SHEET 11 4 LYS A 1016 ASP A  1020  0
SHEET 21 4 TYR A 1024 GLY A  1028  0
SHEET 31 4 HIS A 1031 THR A  1034  0
SHEET 41 4 GLY A 105 THR A  1059  0
SSBOND1 CYS A 106 CYS A  191155515552.05
SSBOND2 CYS A 184 CYS A  190155515552.06
LINKSG CYS A 265C2 ACM A 411155515551.61
LINKSG CYS A 341C1 PLM A 415155515551.62
SITE 1 AC12 ASP A 1072 HOH A 538
SITE 1 AC21 CYS A 265
SITE 1 AC34 LYS A 263 PHE A 264 HIS A 269 HOH A 502
SITE 1 AC54 ASP A 113 PHE A 193 ASN A 312 TYR A 316
SITE 1 AC61 ILE A 112
SITE 1 AC71 HOH A 520
SITE 1 AC97 GLU A 1011 GLY A 1030 LEU A 1032 ASP A 1070
SITE 2 AC97 VAL A 1103 PHE A 1104 ARG A1145
SITE 1 BC12 LEU A 339 CYS A 341
SITE 1 BC25 VAL A 67 THR A 68 ARG A 131 TYR A 141
SITE 2 BC25 SER A 143
SITE 1 BC34 PHE A 264 LYS A 270 LYS A 273 ARG A 328
SITE 1 BC45 PHE A 1114 THR A 1115 ASN A 1116 SER A 1117
SITE 2 BC45 ASN A 1132
SITE 1 BC56 PRO A 1143 ASN A 1144 ARG A 1145 HOH A 512
SITE 2 BC56 HOH A 526 HOH A 531
SITE 1 BC61 ARG A 1095
SITE 1 BC72 LEU A 1015 LYS A 1016
CRYST1106.318 169.240  40.154 90.00 105.62 90.00 C 1 2 1  4
ORIGX11.0000000.0000000.0000000.00000
ORIGX20.0000001.0000000.0000000.00000
ORIGX30.0000000.0000001.0000000.00000
SCALE10.0094060.0000000.0026300.00000
SCALE20.0000000.0059090.0000000.00000
SCALE30.0000000.0000000.0258590.00000
ATOM1NASPA29−52.822−1.61123.1371.0098.48N
ATOM2CAASPA29−51.922−2.26222.1481.0098.06C
ATOM3CASPA29−52.178−1.71320.7421.0097.74C
ATOM4OASPA29−51.291−1.10020.1431.0096.54O
ATOM5CBASPA29−52.106−3.78622.1841.0097.64C
ATOM6NGLUA30−53.394−1.94420.2361.0098.37N
ATOM7CAGLUA30−53.821−1.51518.8871.0098.17C
ATOM8CGLUA30−54.424−0.10418.8791.0098.57C
ATOM9OGLUA30−54.1970.64917.9431.0099.79O
ATOM10CBGLUA30−54.840−2.49818.3051.0099.00C
ATOM11CGGLUA30−54.377−3.96918.2861.0099.46C
ATOM12CDGLUA30−55.432−4.92817.7331.0098.77C
ATOM13OE1GLUA30−56.228−4.52716.8531.0097.60O
ATOM14OE2GLUA30−55.463−6.09218.1851.0099.75O
ATOM15NVALA31−55.1900.24819.9181.0097.89N
ATOM16CAVALA31−55.7571.61820.0791.0096.48C
ATOM17CVALA31−54.6432.67820.1851.0095.12C
ATOM18OVALA31−54.8383.83719.8031.0093.37O
ATOM19CBVALA31−56.7291.69721.3061.0096.71C
ATOM20CG1VALA31−57.1853.13521.5801.0096.80C
ATOM21CG2VALA31−57.9480.80621.0771.0096.80C
ATOM22NTRPA32−53.4862.25920.7091.0095.21N
ATOM23CATRPA32−52.2673.06320.7351.0094.60C
ATOM24CTRPA32−51.8373.49119.3191.0093.60C
ATOM25OTRPA32−51.3354.60319.1401.0091.48O
ATOM26CBTRPA32−51.1292.27321.4271.0096.93C
ATOM27CGTRPA32−49.7702.89921.3041.0098.27C
ATOM28CD1TRPA32−49.1693.73422.1971.00100.48C
ATOM29CD2TRPA32−48.8442.73720.2161.00100.89C
ATOM30NE1TRPA32−47.9244.10521.7371.00101.70N
ATOM31CE2TRPA32−47.7003.50720.5231.00102.25C
ATOM32CE3TRPA32−48.8742.01319.0101.00100.84C
ATOM33CZ2TRPA32−46.5873.57819.6671.00101.34C
ATOM34CZ3TRPA32−47.7712.08218.1581.00100.68C
ATOM35CH2TRPA32−46.6402.86018.4931.00101.20C
ATOM36NVALA33−52.0362.60018.3321.0092.39N
ATOM37CAVALA33−51.6672.85216.9221.0091.10C
ATOM38CVALA33−52.4354.01716.3321.0089.89C
ATOM39OVALA33−51.8274.91815.7481.0088.01O
ATOM40CBVALA33−51.9201.62116.0121.0091.28C
ATOM41CG1VALA33−51.6251.95014.5531.0093.68C
ATOM42CG2VALA33−51.0800.44316.4671.0093.38C
ATOM43NVALA34−53.7623.98616.4901.0088.20N
ATOM44CAVALA34−54.6385.07816.0411.0087.62C
ATOM45CVALA34−54.1456.38816.6491.0087.34C
ATOM46OVALA34−53.8717.33415.9211.0088.42O
ATOM47CBVALA34−56.1264.85216.4221.0087.61C
ATOM48CG1VALA34−56.9726.06416.0461.0087.66C
ATOM49CG2VALA34−56.6843.59215.7551.0085.12C
ATOM50NGLYA35−54.0366.41317.9811.0086.61N
ATOM51CAGLYA35−53.5247.57618.7381.0086.08C
ATOM52CGLYA35−52.1238.04818.3581.0085.30C
ATOM53OGLYA35−51.8379.24518.4081.0085.51O
ATOM54NMETA36−51.2577.11017.9811.0085.55N
ATOM55CAMETA36−49.9197.43117.4861.0084.86C
ATOM56CMETA36−50.0047.88816.0291.0085.06C
ATOM57OMETA36−49.2168.73315.5901.0086.04O
ATOM58CBMETA36−49.0016.21417.5961.0086.69C
ATOM59CGMETA36−47.5096.49617.4001.0088.99C
ATOM60SDMETA36−46.8007.64218.6131.00102.42S
ATOM61CEMETA36−47.2136.85720.1791.00100.39C
ATOM62NGLYA37−50.9597.32415.2871.0083.06N
ATOM63CAGLYA37−51.2277.71313.9111.0082.58C
ATOM64CGLYA37−51.7109.14413.8111.0081.74C
ATOM65OGLYA37−51.1719.91213.0241.0083.08O
ATOM66NILEA38−52.7249.49414.6111.0081.34N
ATOM67CAILEA38−53.26010.86914.6591.0080.88C
ATOM68CILEA38−52.15311.87415.0031.0080.13C
ATOM69OILEA38−52.12112.96414.4531.0081.84O
ATOM70CBILEA38−54.43211.03215.6861.0081.07C
ATOM71CG1ILEA38−55.63510.13215.3561.0081.29C
ATOM72CG2ILEA38−54.91212.47915.7451.0080.45C
ATOM73CD1ILEA38−56.31010.41314.0261.0085.05C
ATOM74NVALA39−51.25511.49615.9111.0079.45N
ATOM75CAVALA39−50.12812.34616.3061.0079.67C
ATOM76CVALA39−49.16612.57515.1541.0079.83C
ATOM77OVALA39−48.80313.71714.8631.0081.09O
ATOM78CBVALA39−49.35211.74017.4961.0081.11C
ATOM79CG1VALA39−47.99612.42517.6711.0078.01C
ATOM80CG2VALA39−50.18511.84318.7701.0081.49C
ATOM81NMETA40−48.75411.49214.5061.0080.50N
ATOM82CAMETA40−47.88111.58913.3201.0080.67C
ATOM83CMETA40−48.57512.33312.1551.0080.62C
ATOM84OMETA40−47.91513.03311.3901.0078.80O
ATOM85CBMETA40−47.40510.19712.8701.0080.79C
ATOM86CGMETA40−46.2949.61313.7451.0080.56C
ATOM87SDMETA40−45.9957.86313.4191.0083.92S
ATOM88CEMETA40−44.4867.58814.3441.0083.67C
ATOM89NSERA41−49.89612.17712.0341.0079.41N
ATOM90CASERA41−50.67212.89811.0201.0079.83C
ATOM91CSERA41−50.65114.40311.2691.0080.21C
ATOM92OSERA41−50.52115.18110.3241.0082.42O
ATOM93CBSERA41−52.10412.39410.9761.0079.47C
ATOM94OGSERA41−52.12811.04410.5611.0082.68O
ATOM95NLEUA42−50.77814.80512.5341.0079.85N
ATOM96CALEUA42−50.67516.21912.9081.0079.83C
ATOM97CLEUA42−49.25616.75212.6871.0078.29C
ATOM98OLEUA42−49.09317.91712.3551.0080.15O
ATOM99CBLEUA42−51.10616.44814.3641.0080.71C
ATOM100CGLEUA42−52.58616.24214.7141.0081.76C
ATOM101CD1LEUA42−52.77416.40016.2131.0083.86C
ATOM102CD2LEUA42−53.50817.19113.9561.0082.80C
ATOM103NILEA43−48.24315.90212.8761.0077.38N
ATOM104CAILEA43−46.85016.27412.5931.0077.38C
ATOM105CILEA43−46.68316.60411.1121.0077.46C
ATOM106OILEA43−46.07217.60810.7711.0077.58O
ATOM107CBILEA43−45.84515.15512.9951.0078.54C
ATOM108CG1ILEA43−45.69815.08014.5201.0080.06C
ATOM109CG2ILEA43−44.47315.38712.3661.0074.64C
ATOM110CD1ILEA43−44.92313.84515.0021.0077.13C
ATOM111NVALA44−47.22915.75010.2471.0077.58N
ATOM112CAVALA44−47.19515.9678.8011.0076.60C
ATOM113CVALA44−47.95117.2518.4161.0078.14C
ATOM114OVALA44−47.44518.0537.6291.0076.84O
ATOM115CBVALA44−47.76914.7558.0411.0076.85C
ATOM116CG1VALA44−47.96715.0796.5671.0075.75C
ATOM117CG2VALA44−46.85613.5338.2191.0070.47C
ATOM118NLEUA45−49.15017.4348.9721.0078.66N
ATOM119CALEUA45−49.95418.6388.7191.0078.89C
ATOM120CLEUA45−49.21919.9019.1631.0079.68C
ATOM121OLEUA45−49.24620.9008.4601.0080.46O
ATOM122CBLEUA45−51.30618.5559.4331.0080.42C
ATOM123CGLEUA45−52.37419.5909.0621.0083.12C
ATOM124CD1LEUA45−52.83519.4147.6161.0086.47C
ATOM125CD2LEUA45−53.55519.48510.0081.0082.85C
ATOM126NALAA46−48.57119.83010.3301.0078.89N
ATOM127CAALAA46−47.78320.93310.8931.0076.32C
ATOM128CALAA46−46.63921.3529.9851.0075.76C
ATOM129OALAA46−46.45322.5399.7311.0078.99O
ATOM130CBALAA46−47.22720.53512.2441.0075.15C
ATOM131NILEA47−45.88520.3719.5051.0072.82N
ATOM132CAILEA47−44.75120.6138.5961.0072.71C
ATOM133CILEA47−45.20621.2167.2671.0069.65C
ATOM134OILEA47−44.65122.2096.8151.0068.44O
ATOM135CBILEA47−43.98419.3108.2901.0073.57C
ATOM136CG1ILEA47−43.27618.7779.5401.0073.61C
ATOM137CG2ILEA47−42.96119.5347.1791.0073.63C
ATOM138CD1ILEA47−42.81117.3589.3991.0071.65C
ATOM139NVALA48−46.21620.6066.6591.0068.32N
ATOM140CAVALA48−46.72121.0515.3601.0069.33C
ATOM141CVALA48−47.39022.4405.4631.0071.00C
ATOM142OVALA48−47.11523.3054.6381.0072.48O
ATOM143CBVALA48−47.69220.0114.7221.0069.55C
ATOM144CG1VALA48−48.17820.4893.3641.0064.85C
ATOM145CG2VALA48−47.00818.6424.5851.0066.28C
ATOM146NPHEA49−48.24822.6406.4681.0070.84N
ATOM147CAPHEA49−48.95123.9166.6541.0069.45C
ATOM148CPHEA49−47.99425.0776.8031.0071.34C
ATOM149OPHEA49−48.03926.0256.0171.0071.00O
ATOM150CBPHEA49−49.86923.8667.8861.0070.88C
ATOM151CGPHEA49−50.66925.1238.1031.0071.45C
ATOM152CD1PHEA49−51.93225.2547.5541.0072.92C
ATOM153CD2PHEA49−50.16126.1768.8551.0075.73C
ATOM154CE1PHEA49−52.67826.4147.7511.0073.41C
ATOM155CE2PHEA49−50.90527.3369.0521.0074.53C
ATOM156CZPHEA49−52.16227.4508.4981.0069.47C
ATOM157NGLYA50−47.13324.9857.8211.0071.43N
ATOM158CAGLYA50−46.19326.0408.1581.0067.82C
ATOM159CGLYA50−45.21026.4097.0661.0068.17C
ATOM160OGLYA50−44.95227.5926.8521.0067.71O
ATOM161NASNA51−44.66225.4026.3811.0066.99N
ATOM162CAASNA51−43.71325.6365.2731.0067.98C
ATOM163CASNA51−44.41426.1453.9981.0071.16C
ATOM164OASNA51−43.84726.9683.2741.0069.41O
ATOM165CBASNA51−42.89024.3954.9971.0068.88C
ATOM166CGASNA51−41.92724.0836.1341.0071.66C
ATOM167OD1ASNA51−40.91424.7586.3051.0069.08O
ATOM168ND2ASNA51−42.24123.0636.9131.0068.08N
ATOM169NVALA52−45.63125.6563.7301.0067.56N
ATOM170CAVALA52−46.45326.2452.6871.0070.68C
ATOM171CVALA52−46.61627.7312.9941.0071.64C
ATOM172OVALA52−46.46328.5582.1081.0072.88O
ATOM173CBVALA52−47.86025.5992.5751.0069.27C
ATOM174CG1VALA52−48.83726.5491.9031.0071.90C
ATOM175CG2VALA52−47.78424.2861.8291.0067.05C
ATOM176NLEUA53−46.92128.0494.2561.0073.15N
ATOM177CALEUA53−47.15629.4414.6881.0072.57C
ATOM178CLEUA53−45.91330.3354.5001.0073.72C
ATOM179OLEUA53−46.04331.4944.1201.0076.55O
ATOM180CBLEUA53−47.63229.4826.1471.0072.34C
ATOM181CGLEUA53−48.42230.7056.5931.0074.13C
ATOM182CD1LEUA53−49.81230.7165.9731.0073.24C
ATOM183CD2LEUA53−48.53030.7388.1091.0076.12C
ATOM184NVALA54−44.72329.7894.7651.0072.68N
ATOM185CAVALA54−43.45830.5174.5631.0070.92C
ATOM186CVALA54−43.18330.7953.0761.0072.46C
ATOM187OVALA54−42.78631.9032.7111.0072.67O
ATOM188CBVALA54−42.25529.7415.1611.0073.28C
ATOM189CG1VALA54−40.93730.3804.7691.0072.16C
ATOM190CG2VALA54−42.37629.6476.6821.0069.63C
ATOM191NILEA55−43.39529.7872.2331.0069.98N
ATOM192CAILEA55−43.16429.9060.7911.0068.58C
ATOM193CILEA55−44.07630.9600.1771.0069.05C
ATOM194OILEA55−43.60631.844−0.5521.0065.40O
ATOM195CBILEA55−43.37928.5690.0901.0068.25C
ATOM196CG1ILEA55−42.25327.5910.4541.0071.45C
ATOM197CG2ILEA55−43.43028.766−1.4091.0072.11C
ATOM198CD1ILEA55−42.57326.1520.1271.0071.51C
ATOM199NTHRA56−45.36930.8400.4911.0070.28N
ATOM200CATHRA56−46.40331.7700.0671.0069.62C
ATOM201CTHRA56−46.07833.2110.4541.0071.19C
ATOM202OTHRA56−46.18834.089−0.3721.0072.89O
ATOM203CBTHRA56−47.78031.3990.6861.0071.36C
ATOM204OG1THRA56−48.08230.0190.4341.0065.43O
ATOM205CG2THRA56−48.89632.2810.1101.0069.44C
ATOM206NALAA57−45.67833.4231.7141.0071.45N
ATOM207CAALAA57−45.36834.7622.2631.0070.84C
ATOM208CALAA57−44.18035.4641.5841.0071.90C
ATOM209OALAA57−44.22036.6651.3391.0070.99O
ATOM210CBALAA57−45.11934.6663.7671.0067.19C
ATOM211NILEA58−43.12834.7121.2891.0071.62N
ATOM212CAILEA58−41.96035.2500.5861.0071.02C
ATOM213CILEA58−42.29435.462−0.9101.0076.54C
ATOM214OILEA58−41.89836.471−1.5031.0079.19O
ATOM215CBILEA58−40.75834.3210.7451.0069.83C
ATOM216CG1ILEA58−40.34334.2352.2271.0069.16C
ATOM217CG2ILEA58−39.58534.805−0.0821.0069.14C
ATOM218CD1ILEA58−39.37133.1292.5131.0070.35C
ATOM219NALAA59−43.01634.511−1.5051.0074.84N
ATOM220CAALAA59−43.42034.599−2.9131.0075.72C
ATOM221CALAA59−44.42235.722−3.1741.0076.56C
ATOM222OALAA59−44.41636.311−4.2561.0077.62O
ATOM223CBALAA59−43.99933.268−3.3851.0072.21C
ATOM224NLYSA60−45.27136.012−2.1841.0076.98N
ATOM225CALYSA60−46.33637.010−2.3231.0076.24C
ATOM226CLYSA60−45.93238.438−1.9651.0077.94C
ATOM227OLYSA60−46.28239.371−2.6941.0075.40O
ATOM228CBLYSA60−47.53236.605−1.4661.0075.25C
ATOM229CGLYSA60−48.66737.578−1.5301.0077.75C
ATOM230CDLYSA60−49.95936.950−1.1641.0078.52C
ATOM231CELYSA60−51.02837.983−1.1891.0079.74C
ATOM232NZLYSA60−52.29937.333−1.0811.0083.19N
ATOM233NPHEA61−45.20938.597−0.8561.0078.18N
ATOM234CAPHEA61−44.86139.919−0.3341.0079.67C
ATOM235CPHEA61−43.45740.379−0.7431.0081.98C
ATOM236OPHEA61−42.44339.855−0.2531.0078.47O
ATOM237CBPHEA61−45.05539.9431.1851.0078.50C
ATOM238CGPHEA61−46.48839.7441.5891.0077.03C
ATOM239CD1PHEA61−46.94638.5132.0131.0074.37C
ATOM240CD2PHEA61−47.38540.7991.5361.0079.08C
ATOM241CE1PHEA61−48.26738.3342.3801.0077.21C
ATOM242CE2PHEA61−48.70840.6211.9031.0077.13C
ATOM243CZPHEA61−49.14139.3822.3261.0075.36C
ATOM244NGLUA62−43.45041.369−1.6481.0084.43N
ATOM245CAGLUA62−42.24742.031−2.2081.0086.75C
ATOM246CGLUA62−41.25642.491−1.1221.0089.37C
ATOM247OGLUA62−40.04042.414−1.3131.0090.84O
ATOM248CBGLUA62−42.69943.219−3.0801.0086.58C
ATOM249CGGLUA62−41.64143.860−3.9671.0087.94C
ATOM250CDGLUA62−42.23444.940−4.9111.0090.76C
ATOM251OE1GLUA62−43.32144.720−5.5051.0096.67O
ATOM252OE2GLUA62−41.61146.016−5.0651.0089.94O
ATOM253NARGA63−41.79842.9640.0031.0090.60N
ATOM254CAARGA63−41.03643.3181.2071.0088.93C
ATOM255CARGA63−40.13942.1771.7051.0088.02C
ATOM256OARGA63−39.00742.4092.1311.0088.49O
ATOM257CBARGA63−42.02843.6912.3091.0091.49C
ATOM258CGARGA63−41.44343.9803.6981.0095.03C
ATOM259CDARGA63−42.57344.3404.6431.0099.84C
ATOM260NEARGA63−42.11344.8415.9391.00103.70N
ATOM261CZARGA63−42.90345.3736.8821.00105.87C
ATOM262NH1ARGA63−44.23145.4916.7081.00107.12N
ATOM263NH2ARGA63−42.36245.7968.0281.00109.62N
ATOM264NLEUA64−40.65840.9551.6481.0085.74N
ATOM265CALEUA64−39.93239.7752.0971.0082.11C
ATOM266CLEUA64−39.03239.1681.0111.0081.55C
ATOM267OLEUA64−38.37838.1841.2721.0078.38O
ATOM268CBLEUA64−40.92338.7122.6021.0080.82C
ATOM269CGLEUA64−41.85139.0903.7691.0079.80C
ATOM270CD1LEUA64−42.89937.9994.0071.0070.96C
ATOM271CD2LEUA64−41.05639.3375.0381.0073.56C
ATOM272NGLNA65−38.99239.745−0.1981.0082.04N
ATOM273CAGLNA65−38.17439.189−1.3081.0083.43C
ATOM274CGLNA65−36.72039.672−1.2641.0083.31C
ATOM275OGLNA65−36.30540.518−2.0601.0086.84O
ATOM276CBGLNA65−38.82039.500−2.6621.0082.54C
ATOM277CGGLNA65−40.12038.749−2.8561.0084.91C
ATOM278CDGLNA65−40.78939.016−4.1821.0088.24C
ATOM279OE1GLNA65−40.51640.018−4.8571.0097.81O
ATOM280NE2GLNA65−41.68138.114−4.5691.0097.75N
ATOM281NTHRA66−35.97339.113−0.3201.0080.37N
ATOM282CATHRA66−34.56139.413−0.0901.0079.80C
ATOM283CTHRA66−33.74738.170−0.3551.0079.26C
ATOM284OTHRA66−34.30637.105−0.4281.0082.12O
ATOM285CBTHRA66−34.33439.7781.3531.0079.66C
ATOM286OG1THRA66−34.64138.6392.1771.0079.38O
ATOM287CG2THRA66−35.21840.9531.7531.0075.58C
ATOM288NVALA67−32.43138.303−0.4921.0076.86N
ATOM289CAVALA67−31.56137.138−0.7711.0076.42C
ATOM290CVALA67−31.67936.0800.3421.0076.58C
ATOM291OVALA67−31.86934.8880.0501.0076.26O
ATOM292CBVALA67−30.08737.543−0.9561.0077.31C
ATOM293CG1VALA67−29.18536.325−0.9141.0075.37C
ATOM294CG2VALA67−29.90038.301−2.2701.0072.45C
ATOM295NTHRA68−31.56836.5121.5971.0071.98N
ATOM296CATHRA68−31.75435.6092.7361.0072.07C
ATOM297CTHRA68−33.02834.7972.5661.0071.33C
ATOM298OTHRA68−33.00133.5642.6451.0069.01O
ATOM299CBTHRA68−31.81036.3724.0621.0071.58C
ATOM300OG1THRA68−30.54637.0094.2831.0073.28O
ATOM301CG2THRA68−32.09835.4205.2131.0071.59C
ATOM302NASNA69−34.13935.4882.3211.0071.44N
ATOM303CAASNA69−35.43834.8102.1461.0070.87C
ATOM304CASNA69−35.53433.8420.9571.0071.14C
ATOM305OASNA69−36.42532.9810.9411.0073.32O
ATOM306CBASNA69−36.57135.8352.1231.0068.01C
ATOM307CGASNA69−36.79436.5013.5031.0072.26C
ATOM308OD1ASNA69−36.31136.0184.5391.0066.06O
ATOM309ND2ASNA69−37.52537.6063.5131.0072.74N
ATOM310NTYRA70−34.64033.969−0.0231.0072.21N
ATOM311CATYRA70−34.57833.002−1.1331.0073.01C
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ATOM922CGLYSA147−33.10739.543−6.2641.0081.83C
ATOM923CDLYSA147−33.37739.263−4.8151.0087.01C
ATOM924CELYSA147−33.75840.559−4.0721.0089.91C
ATOM925NZLYSA147−35.04841.155−4.5561.0091.41N
ATOM926NASNA148−30.51236.843−8.8431.0076.47N
ATOM927CAASNA148−30.19935.809−9.8491.0077.14C
ATOM928CASNA148−29.02334.912−9.4761.0076.20C
ATOM929OASNA148−29.11333.689−9.6471.0074.96O
ATOM930CBASNA148−29.95836.431−11.2441.0079.53C
ATOM931CGASNA148−31.25436.824−11.9561.0084.32C
ATOM932OD1ASNA148−32.35536.395−11.5861.0089.19O
ATOM933ND2ASNA148−31.12137.648−12.9931.0085.88N
ATOM934NLYSA149−27.93335.503−8.9761.0073.79N
ATOM935CALYSA149−26.80134.706−8.4741.0076.59C
ATOM936CLYSA149−27.25433.809−7.3191.0076.35C
ATOM937OLYSA149−26.82232.672−7.2321.0081.05O
ATOM938CBLYSA149−25.66235.562−7.9501.0079.46C
ATOM939CGLYSA149−25.00736.526−8.9141.0082.58C
ATOM940CDLYSA149−24.00137.409−8.1291.0083.85C
ATOM941CELYSA149−23.69038.747−8.7901.0089.02C
ATOM942NZLYSA149−23.01439.645−7.7731.0088.17N
ATOM943NALAA150−28.12034.331−6.4411.0073.36N
ATOM944CAALAA150−28.68433.545−5.3251.0074.46C
ATOM945CALAA150−29.45732.309−5.8271.0075.42C
ATOM946OALAA150−29.33731.239−5.2451.0079.11O
ATOM947CBALAA150−29.57234.416−4.4401.0068.50C
ATOM948NARGA151−30.24332.465−6.9001.0077.55N
ATOM949CAARGA151−30.94531.314−7.5111.0077.51C
ATOM950CARGA151−29.98330.249−8.0221.0077.61C
ATOM951OARGA151−30.27229.057−7.9131.0077.15O
ATOM952CBARGA151−31.83631.709−8.6901.0083.00C
ATOM953CGARGA151−33.28432.095−8.3761.0089.58C
ATOM954CDARGA151−33.57633.556−8.5981.0095.56C
ATOM955NEARGA151−34.99033.794−8.8841.0093.67N
ATOM956CZARGA151−35.51034.981−9.2201.0098.85C
ATOM957NH1ARGA151−34.74336.084−9.3251.0097.06N
ATOM958NH2ARGA151−36.82035.073−9.4581.00102.25N
ATOM959NVALA152−28.85230.684−8.5791.0077.11N
ATOM960CAVALA152−27.82529.767−9.0811.0077.01C
ATOM961CVALA152−27.14429.030−7.9191.0076.31C
ATOM962OVALA152−26.87827.845−8.0231.0073.81O
ATOM963CBVALA152−26.80630.502−9.9871.0078.53C
ATOM964CG1VALA152−25.61229.617−10.3111.0085.33C
ATOM965CG2VALA152−27.48630.934−11.2741.0075.42C
ATOM966NILEA153−26.87429.734−6.8241.0076.01N
ATOM967CAILEA153−26.27829.102−5.6321.0076.47C
ATOM968CILEA153−27.23528.053−5.0741.0075.66C
ATOM969OILEA153−26.81226.945−4.7331.0074.99O
ATOM970CBILEA153−25.95430.101−4.5231.0075.93C
ATOM971CG1ILEA153−24.89931.127−4.9661.0078.94C
ATOM972CG2ILEA153−25.40229.373−3.3201.0075.34C
ATOM973CD1ILEA153−23.48830.564−5.0631.0084.91C
ATOM974NILEA154−28.51728.416−4.9901.0075.41N
ATOM975CAILEA154−29.57027.497−4.5451.0075.93C
ATOM976CILEA154−29.64526.286−5.4611.0072.61C
ATOM977OILEA154−29.56525.159−5.0011.0070.17O
ATOM978CBILEA154−30.94328.192−4.4901.0075.92C
ATOM979CG1ILEA154−30.97529.183−3.3281.0079.46C
ATOM980CG2ILEA154−32.07227.170−4.3121.0073.88C
ATOM981CD1ILEA154−32.13030.133−3.3711.0081.26C
ATOM982NLEUA155−29.79326.521−6.7541.0075.14N
ATOM983CALEUA155−29.82625.411−7.7251.0075.92C
ATOM984CLEUA155−28.60424.497−7.5431.0075.22C
ATOM985OLEUA155−28.74423.279−7.5431.0073.02O
ATOM986CBLEUA155−29.91225.930−9.1581.0071.84C
ATOM987CGLEUA155−29.83724.903−10.2971.0079.20C
ATOM988CD1LEUA155−30.84523.769−10.0951.0082.92C
ATOM989CD2LEUA155−30.03825.585−11.6661.0075.89C
ATOM990NMETA156−27.42325.100−7.3841.0076.89N
ATOM991CAMETA156−26.18624.339−7.1841.0077.43C
ATOM992CMETA156−26.18123.568−5.8711.0076.37C
ATOM993OMETA156−25.57122.504−5.8011.0078.59O
ATOM994CBMETA156−24.94525.247−7.2811.0081.89C
ATOM995CGMETA156−24.60925.744−8.7161.0083.04C
ATOM996SDMETA156−24.28424.442−9.9421.00100.97S
ATOM997CEMETA156−24.12825.398−11.4581.0089.68C
ATOM998NVALA157−26.85124.094−4.8411.0076.00N
ATOM999CAVALA157−27.00023.370−3.5721.0074.39C
ATOM1000CVALA157−27.83522.098−3.7841.0074.09C
ATOM1001OVALA157−27.44721.022−3.3271.0074.06O
ATOM1002CBVALA157−27.62424.250−2.4481.0073.43C
ATOM1003CG1VALA157−28.15223.382−1.3211.0069.33C
ATOM1004CG2VALA157−26.61325.269−1.9421.0068.63C
ATOM1005NTRPA158−28.96922.227−4.4721.0074.10N
ATOM1006CATRPA158−29.81721.046−4.7771.0074.40C
ATOM1007CTRPA158−29.13120.024−5.6831.0075.32C
ATOM1008OTRPA158−29.31318.824−5.4821.0078.30O
ATOM1009CBTRPA158−31.18821.469−5.3101.0070.20C
ATOM1010CGTRPA158−31.96121.950−4.1831.0074.02C
ATOM1011CD1TRPA158−32.01523.217−3.7191.0080.00C
ATOM1012CD2TRPA158−32.80321.173−3.3331.0072.24C
ATOM1013NE1TRPA158−32.84223.290−2.6321.0079.53N
ATOM1014CE2TRPA158−33.34222.050−2.3711.0079.45C
ATOM1015CE3TRPA158−33.15719.822−3.2921.0073.80C
ATOM1016CZ2TRPA158−34.22021.625−1.3741.0071.01C
ATOM1017CZ3TRPA158−34.02619.396−2.3071.0073.95C
ATOM1018CH2TRPA158−34.54920.299−1.3581.0074.98C
ATOM1019NILEA159−28.35420.495−6.6631.0076.28N
ATOM1020CAILEA159−27.57219.595−7.5261.0075.87C
ATOM1021CILEA159−26.50918.840−6.7131.0076.85C
ATOM1022OILEA159−26.41617.626−6.8301.0076.71O
ATOM1023CBILEA159−26.89220.332−8.6961.0073.88C
ATOM1024CG1ILEA159−27.93320.757−9.7351.0075.94C
ATOM1025CG2ILEA159−25.86119.431−9.3541.0076.05C
ATOM1026CD1ILEA159−27.35921.529−10.9231.0071.99C
ATOM1027NVALA160−25.72519.564−5.9031.0074.32N
ATOM1028CAVALA160−24.67218.947−5.0731.0074.37C
ATOM1029CVALA160−25.25717.970−4.0471.0076.12C
ATOM1030OVALA160−24.73816.864−3.8771.0072.86O
ATOM1031CBVALA160−23.80520.011−4.3571.0075.98C
ATOM1032CG1VALA160−22.91019.378−3.2971.0070.80C
ATOM1033CG2VALA160−22.96220.777−5.3771.0072.65C
ATOM1034NSERA161−26.33218.388−3.3761.0076.74N
ATOM1035CASERA161−27.05217.522−2.4361.0076.33C
ATOM1036CSERA161−27.58816.253−3.1271.0080.12C
ATOM1037OSERA161−27.56515.175−2.5401.0081.12O
ATOM1038CBSERA161−28.20318.277−1.7831.0077.19C
ATOM1039OGSERA161−27.72119.391−1.0431.0076.25O
ATOM1040NGLYA162−28.06716.399−4.3671.0078.97N
ATOM1041CAGLYA162−28.55915.268−5.1651.0077.45C
ATOM1042CGLYA162−27.41814.346−5.5591.0075.17C
ATOM1043OGLYA162−27.53613.134−5.4951.0072.75O
ATOM1044NLEUA163−26.31314.958−5.9651.0076.45N
ATOM1045CALEUA163−25.09814.253−6.3551.0078.44C
ATOM1046CLEUA163−24.51713.473−5.1711.0079.50C
ATOM1047OLEUA163−23.96212.407−5.3561.0080.46O
ATOM1048CBLEUA163−24.04415.248−6.9081.0079.15C
ATOM1049CGLEUA163−23.15314.848−8.0811.0079.51C
ATOM1050CD1LEUA163−23.97814.520−9.3081.0075.74C
ATOM1051CD2LEUA163−22.18315.984−8.3861.0078.31C
ATOM1052NTHRA164−24.65014.007−3.9581.0082.19N
ATOM1053CATHRA164−24.15013.328−2.7511.0084.31C
ATOM1054CTHRA164−25.19612.399−2.0611.0085.47C
ATOM1055OTHRA164−24.81411.585−1.2251.0083.60O
ATOM1056CBTHRA164−23.56014.350−1.7581.0084.63C
ATOM1057OG1THRA164−24.50015.405−1.5141.0086.96O
ATOM1058CG2THRA164−22.29114.950−2.3371.0082.27C
ATOM1059NSERA165−26.48512.525−2.4151.0087.63N
ATOM1060CASERA165−27.56211.705−1.8321.0088.01C
ATOM1061CSERA165−28.05710.621−2.7781.0090.10C
ATOM1062OSERA165−28.0299.438−2.4321.0094.95O
ATOM1063CBSERA165−28.74512.583−1.4541.0092.00C
ATOM1064OGSERA165−29.32413.166−2.6141.0096.93O
ATOM1065NPHEA166−28.51211.027−3.9671.0087.60N
ATOM1066CAPHEA166−29.07810.078−4.9511.0085.74C
ATOM1067CPHEA166−28.0429.150−5.5731.0085.31C
ATOM1068OPHEA166−28.3137.967−5.7041.0085.33O
ATOM1069CBPHEA166−29.88410.789−6.0731.0085.68C
ATOM1070CGPHEA166−31.35010.954−5.7531.0084.00C
ATOM1071CD1PHEA166−32.30710.148−6.3621.0082.71C
ATOM1072CD2PHEA166−31.77311.910−4.8471.0078.83C
ATOM1073CE1PHEA166−33.65910.302−6.0661.0084.17C
ATOM1074CE2PHEA166−33.12412.065−4.5511.0079.25C
ATOM1075CZPHEA166−34.06211.265−5.1551.0080.39C
ATOM1076NLEUA167−26.8709.673−5.9541.0083.84N
ATOM1077CALEUA167−25.8398.813−6.5791.0083.08C
ATOM1078CLEUA167−25.5107.568−5.7381.0083.34C
ATOM1079OLEUA167−25.7256.477−6.2351.0084.70O
ATOM1080CBLEUA167−24.5439.568−6.9471.0082.52C
ATOM1081CGLEUA167−24.43110.342−8.2501.0081.28C
ATOM1082CD1LEUA167−23.00310.847−8.4151.0076.32C
ATOM1083CD2LEUA167−24.8159.492−9.4271.0075.15C
ATOM1084NPROA168−25.0037.727−4.4801.0082.18N
ATOM1085CAPROA168−24.6576.540−3.6631.0081.22C
ATOM1086CPROA168−25.8035.561−3.4191.0080.15C
ATOM1087OPROA168−25.5784.352−3.4391.0079.49O
ATOM1088CBPROA168−24.2267.145−2.3211.0080.06C
ATOM1089CGPROA168−23.8588.515−2.6141.0082.53C
ATOM1090CDPROA168−24.7208.968−3.7341.0083.35C
ATOM1091NILEA169−27.0076.086−3.1911.0080.34N
ATOM1092CAILEA169−28.1815.246−2.9311.0081.83C
ATOM1093CILEA169−28.6704.542−4.2071.0083.09C
ATOM1094OILEA169−28.9743.347−4.1671.0085.69O
ATOM1095CBILEA169−29.3166.029−2.2451.0080.40C
ATOM1096CG1ILEA169−28.8456.522−0.8761.0082.52C
ATOM1097CG2ILEA169−30.5385.150−2.0691.0080.48C
ATOM1098CD1ILEA169−29.9137.192−0.0591.0083.50C
ATOM1099NGLNA170−28.7435.274−5.3221.0083.76N
ATOM1100CAGLNA170−29.1614.691−6.6171.0083.63C
ATOM1101CGLNA170−28.0563.817−7.2431.0083.39C
ATOM1102OGLNA170−28.3742.864−7.9601.0086.11O
ATOM1103CBGLNA170−29.6065.780−7.6161.0084.12C
ATOM1104CGGLNA170−30.7666.740−7.1231.0084.07C
ATOM1105CDGLNA170−32.1596.134−7.1191.0080.98C
ATOM1106OE1GLNA170−32.3705.018−7.5571.0077.62O
ATOM1107NE2GLNA170−33.1236.895−6.6141.0076.26N
ATOM1108NMETA171−26.7774.134−6.9811.0082.82N
ATOM1109CAMETA171−25.6363.286−7.4361.0083.78C
ATOM1110CMETA171−25.3402.113−6.4931.0083.40C
ATOM1111OMETA171−24.4871.279−6.8161.0079.60O
ATOM1112CBMETA171−24.3364.104−7.6311.0084.08C
ATOM1113CGMETA171−24.3155.014−8.8541.0084.82C
ATOM1114SDMETA171−24.3724.141−10.4451.0093.60S
ATOM1115CEMETA171−22.9623.018−10.3561.0086.88C
ATOM1116NHISA172−26.0322.056−5.3461.0083.18N
ATOM1117CAHISA172−25.9090.954−4.3731.0083.41C
ATOM1118CHISA172−24.5440.943−3.6211.0083.06C
ATOM1119OHISA172−24.082−0.107−3.1681.0083.35O
ATOM1120CBHISA172−26.218−0.415−5.0501.0082.96C
ATOM1121CGHISA172−27.640−0.563−5.5051.0083.78C
ATOM1122ND1HISA172−28.273−1.780−5.5171.0079.73N
ATOM1123CD2HISA172−28.5520.332−5.9571.0082.46C
ATOM1124CE1HISA172−29.509−1.639−5.9541.0077.50C
ATOM1125NE2HISA172−29.705−0.364−6.2291.0081.47N
ATOM1126NTRPA173−23.9152.116−3.4951.0083.44N
ATOM1127CATRPA173−22.6472.253−2.7581.0083.86C
ATOM1128CTRPA173−22.8232.178−1.2441.0083.60C
ATOM1129OTRPA173−21.8881.833−0.5201.0083.83O
ATOM1130CBTRPA173−21.9533.576−3.0841.0083.86C
ATOM1131CGTRPA173−21.4923.720−4.4911.0084.03C
ATOM1132CD1TRPA173−21.4962.766−5.4751.0084.91C
ATOM1133CD2TRPA173−20.9394.893−5.0791.0082.79C
ATOM1134NE1TRPA173−20.9883.281−6.6311.0084.59N
ATOM1135CE2TRPA173−20.6364.585−6.4191.0081.34C
ATOM1136CE3TRPA173−20.6716.184−4.6021.0084.75C
ATOM1137CZ2TRPA173−20.0795.517−7.2911.0085.31C
ATOM1138CZ3TRPA173−20.1157.115−5.4701.0084.09C
ATOM1139CH2TRPA173−19.8256.777−6.7991.0086.15C
ATOM1140NTYRA174−24.0182.505−0.7771.0081.87N
ATOM1141CATYRA174−24.3302.4780.6431.0082.95C
ATOM1142CTYRA174−24.4611.1031.2611.0084.39C
ATOM1143OTYRA174−24.3280.9932.4511.0085.16O
ATOM1144CBTYRA174−25.6463.2240.9161.0082.89C
ATOM1145CGTYRA174−26.9132.4730.5221.0080.98C
ATOM1146CD1TYRA174−27.6791.7941.4751.0079.89C
ATOM1147CD2TYRA174−27.3402.442−0.7981.0079.78C
ATOM1148CE1TYRA174−28.8381.1101.1111.0081.50C
ATOM1149CE2TYRA174−28.4891.767−1.1741.0080.48C
ATOM1150CZTYRA174−29.2341.104−0.2211.0081.26C
ATOM1151OHTYRA174−30.3650.439−0.6011.0079.71O
ATOM1152NARGA175−24.7090.0700.4571.0082.91N
ATOM1153CAARGA175−25.158−1.2270.9841.0081.71C
ATOM1154CARGA175−24.093−2.0471.6781.0080.96C
ATOM1155OARGA175−22.922−1.9981.3131.0080.38O
ATOM1156CBARGA175−25.819−2.057−0.1361.0082.53C
ATOM1157CGARGA175−26.861−1.242−0.8631.0085.26C
ATOM1158CDARGA175−28.068−1.948−1.3181.0083.28C
ATOM1159NEARGA175−27.867−2.850−2.4131.0082.86N
ATOM1160CZARGA175−28.850−3.346−3.1721.0086.74C
ATOM1161NH1ARGA175−30.142−3.019−2.9631.0084.23N
ATOM1162NH2ARGA175−28.541−4.184−4.1691.0081.76N
ATOM1163NALAA176−24.539−2.7932.6891.0081.02N
ATOM1164CAALAA176−23.696−3.6873.4541.0081.16C
ATOM1165CALAA176−23.664−5.0202.7371.0081.72C
ATOM1166OALAA176−24.541−5.3081.9201.0081.33O
ATOM1167CBALAA176−24.231−3.8534.8541.0081.01C
ATOM1168NTHRA177−22.651−5.8213.0561.0081.43N
ATOM1169CATHRA177−22.421−7.1282.4221.0080.81C
ATOM1170CTHRA177−22.741−8.3483.3191.0081.25C
ATOM1171OTHRA177−22.430−9.4812.9471.0081.14O
ATOM1172CBTHRA177−20.959−7.2151.9441.0080.88C
ATOM1173OG1THRA177−20.080−7.1113.0711.0080.59O
ATOM1174CG2THRA177−20.656−6.0900.9611.0080.78C
ATOM1175NHISA178−23.358−8.1234.4821.0080.75N
ATOM1176CAHISA178−23.719−9.2275.3971.0080.59C
ATOM1177CHISA178−25.129−9.7235.0471.0079.62C
ATOM1178OHISA178−25.924−8.9854.4731.0080.66O
ATOM1179CBHISA178−23.574−8.8246.8781.0080.59C
ATOM1180CGHISA178−24.512−7.7527.3201.0080.24C
ATOM1181ND1HISA178−24.229−6.4107.1891.0085.34N
ATOM1182CD2HISA178−25.731−7.8257.8951.0081.58C
ATOM1183CE1HISA178−25.238−5.7027.6641.0086.20C
ATOM1184NE2HISA178−26.163−6.5378.0981.0088.13N
ATOM1185NGLNA179−25.413−10.9735.4051.0079.51N
ATOM1186CAGLNA179−26.652−11.6704.9941.0079.05C
ATOM1187CGLNA179−27.968−11.0245.4531.0077.54C
ATOM1188OGLNA179−28.903−10.9374.6671.0076.07O
ATOM1189CBGLNA179−26.624−13.1405.4671.0079.94C
ATOM1190CGGLNA179−27.540−14.0794.6681.0082.48C
ATOM1191CDGLNA179−27.127−14.2183.2011.0085.58C
ATOM1192OE1GLNA179−25.963−14.0062.8381.0084.07O
ATOM1193NE2GLNA179−28.082−14.5782.3551.0088.48N
ATOM1194NGLUA180−28.026−10.5876.7131.0076.40N
ATOM1195CAGLUA180−29.224−9.9157.2781.0076.08C
ATOM1196CGLUA180−29.656−8.6946.4411.0074.97C
ATOM1197OGLUA180−30.854−8.4376.2681.0072.77O
ATOM1198CBGLUA180−28.957−9.5188.7381.0077.18C
ATOM1199CGGLUA180−30.098−8.8299.4991.0078.62C
ATOM1200CDGLUA180−29.685−8.43010.9201.0078.93C
ATOM1201OE1GLUA180−30.024−7.30311.3361.0077.22O
ATOM1202OE2GLUA180−29.022−9.23911.6181.0082.04O
ATOM1203NALAA181−28.666−7.9625.9291.0073.84N
ATOM1204CAALAA181−28.887−6.8145.0611.0074.58C
ATOM1205CALAA181−29.359−7.2913.6991.0074.81C
ATOM1206OALAA181−30.389−6.8283.2011.0074.62O
ATOM1207CBALAA181−27.610−6.0054.9211.0072.34C
ATOM1208NILEA182−28.592−8.2183.1101.0076.12N
ATOM1209CAILEA182−28.905−8.8431.8001.0076.87C
ATOM1210CILEA182−30.356−9.3361.7431.0077.84C
ATOM1211OILEA182−31.047−9.1090.7451.0078.53O
ATOM1212CBILEA182−27.924−10.0031.4531.0076.51C
ATOM1213CG1ILEA182−26.515−9.4661.1921.0075.02C
ATOM1214CG2ILEA182−28.387−10.7560.2261.0076.30C
ATOM1215CD1ILEA182−25.478−10.5400.9591.0075.60C
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ATOM1218CASNA183−33.152−9.2632.9241.0081.18C
ATOM1219OASNA183−34.160−9.3232.2321.0081.85O
ATOM1220CBASNA183−32.392−11.2254.2601.0079.70C
ATOM1221CGASNA183−31.725−12.5864.2461.0078.69C
ATOM1222OD1ASNA183−30.711−12.8003.5861.0076.53O
ATOM1223ND2ASNA183−32.301−13.5224.9841.0078.92N
ATOM1224NCYSA184−32.835−8.2003.6711.0081.90N
ATOM1225CACYSA184−33.675−6.9903.6981.0081.53C
ATOM1226CCYSA184−33.742−6.2782.3481.0080.40C
ATOM1227OCYSA184−34.791−5.7431.9821.0080.62O
ATOM1228CBCYSA184−33.189−5.9954.7511.0082.03C
ATOM1229SGCYSA184−34.281−4.5564.8961.0086.09S
ATOM1230NTYRA185−32.630−6.2701.6141.0080.51N
ATOM1231CATYRA185−32.588−5.6260.2891.0080.30C
ATOM1232CTYRA185−33.500−6.328−0.6931.0080.59C
ATOM1233OTYRA185−34.322−5.683−1.3541.0082.14O
ATOM1234CBTYRA185−31.165−5.597−0.2781.0079.80C
ATOM1235CGTYRA185−30.177−4.7980.5521.0080.24C
ATOM1236CD1TYRA185−30.497−3.5221.0181.0076.86C
ATOM1237CD2TYRA185−28.923−5.3130.8661.0079.73C
ATOM1238CE1TYRA185−29.604−2.7971.7691.0078.10C
ATOM1239CE2TYRA185−28.021−4.5891.6181.0079.03C
ATOM1240CZTYRA185−28.365−3.3342.0661.0078.33C
ATOM1241OHTYRA185−27.471−2.6212.8111.0079.23O
ATOM1242NALAA186−33.344−7.645−0.7761.0079.03N
ATOM1243CAALAA186−34.189−8.489−1.6221.0078.43C
ATOM1244CALAA186−35.659−8.474−1.1781.0078.71C
ATOM1245OALAA186−36.557−8.506−2.0231.0079.49O
ATOM1246CBALAA186−33.661−9.914−1.6271.0076.05C
ATOM1247NGLUA187−35.887−8.4220.1401.0079.25N
ATOM1248CAGLUA187−37.237−8.3990.7131.0079.62C
ATOM1249CGLUA187−37.976−7.0850.4001.0079.69C
ATOM1250OGLUA187−37.549−5.9980.8141.0080.47O
ATOM1251CBGLUA187−37.181−8.6432.2341.0079.95C
ATOM1252CGGLUA187−38.523−8.6172.9921.0082.26C
ATOM1253CDGLUA187−39.521−9.6712.5331.0086.24C
ATOM1254OE1GLUA187−39.137−10.6431.8451.0092.89O
ATOM1255OE2GLUA187−40.711−9.5302.8631.0088.95O
ATOM1256NGLUA188−39.081−7.216−0.3341.0079.07N
ATOM1257CAGLUA188−39.930−6.085−0.7511.0079.55C
ATOM1258CGLUA188−40.619−5.3610.4041.0079.89C
ATOM1259OGLUA188−40.897−4.1610.2921.0079.15O
ATOM1260CBGLUA188−40.983−6.562−1.7471.0079.62C
ATOM1261CGGLUA188−42.048−7.458−1.1211.0083.66C
ATOM1262CDGLUA188−42.614−8.463−2.0841.0084.99C
ATOM1263OE1GLUA188−42.812−8.122−3.2681.0095.21O
ATOM1264OE2GLUA188−42.866−9.605−1.6521.0089.59O
ATOM1265NTHRA189−40.891−6.0891.4961.0077.96N
ATOM1266CATHRA189−41.533−5.5122.6761.0075.91C
ATOM1267CTHRA189−40.541−5.0633.7551.0076.43C
ATOM1268OTHRA189−40.961−4.6564.8361.0076.97O
ATOM1269CBTHRA189−42.562−6.4813.3001.0075.78C
ATOM1270OG1THRA189−41.883−7.6173.8431.0074.95O
ATOM1271CG2THRA189−43.580−6.9232.2521.0070.98C
ATOM1272NCYSA190−39.242−5.1313.4641.0078.00N
ATOM1273CACYSA190−38.209−4.6904.3771.0077.37C
ATOM1274CCYSA190−37.588−3.4433.7991.0074.95C
ATOM1275OCYSA190−37.131−3.4642.6581.0075.26O
ATOM1276CBCYSA190−37.136−5.7554.5451.0079.47C
ATOM1277SGCYSA190−35.924−5.3475.8471.0085.35S
ATOM1278NCYSA191−37.574−2.3684.5821.0075.05N
ATOM1279CACYSA191−36.955−1.1104.1751.0077.17C
ATOM1280CCYSA191−36.140−0.5375.3291.0077.29C
ATOM1281OCYSA191−36.4500.5235.8561.0081.42O
ATOM1282CBCYSA191−38.024−0.1353.6801.0077.85C
ATOM1283SGCYSA191−37.3831.3702.8991.0080.61S
ATOM1284NASPA192−35.091−1.2705.7031.0078.03N
ATOM1285CAASPA192−34.141−0.8516.7411.0076.97C
ATOM1286CASPA192−32.909−0.3476.0541.0075.94C
ATOM1287OASPA192−32.428−0.9795.1141.0077.63O
ATOM1288CBASPA192−33.762−2.0057.6671.0077.44C
ATOM1289CGASPA192−34.889−2.4198.5861.0080.00C
ATOM1290OD1ASPA192−35.724−1.5618.9591.0082.18O
ATOM1291OD2ASPA192−34.941−3.6198.9431.0080.09O
ATOM1292NPHEA193−32.3880.7856.5171.0075.48N
ATOM1293CAPHEA193−31.2371.4165.8901.0075.22C
ATOM1294CPHEA193−29.9090.8656.4431.0075.43C
ATOM1295OPHEA193−29.1601.5737.1331.0073.54O
ATOM1296CBPHEA193−31.3512.9476.0431.0074.11C
ATOM1297CGPHEA193−30.3993.7305.1751.0076.00C
ATOM1298CD1PHEA193−30.3003.4713.7931.0076.25C
ATOM1299CD2PHEA193−29.6004.7365.7241.0076.96C
ATOM1300CE1PHEA193−29.4284.1892.9841.0073.80C
ATOM1301CE2PHEA193−28.7185.4654.9051.0079.72C
ATOM1302CZPHEA193−28.6385.1833.5281.0075.51C
ATOM1303NPHEA194−29.641−0.4096.1271.0074.51N
ATOM1304CAPHEA194−28.376−1.0616.4551.0074.06C
ATOM1305CPHEA194−27.340−0.4935.5271.0074.74C
ATOM1306OPHEA194−27.542−0.5224.3081.0073.83O
ATOM1307CBPHEA194−28.423−2.5776.2351.0073.04C
ATOM1308CGPHEA194−29.226−3.3187.2561.0073.26C
ATOM1309CD1PHEA194−28.674−3.6308.4991.0075.81C
ATOM1310CD2PHEA194−30.534−3.7126.9841.0074.89C
ATOM1311CE1PHEA194−29.412−4.3209.4571.0074.15C
ATOM1312CE2PHEA194−31.282−4.4057.9361.0075.40C
ATOM1313CZPHEA194−30.720−4.7099.1761.0074.86C
ATOM1314NTHRA195−26.2410.0226.0871.0073.91N
ATOM1315CATHRA195−25.1570.5595.2781.0072.89C
ATOM1316CTHRA195−23.811−0.0395.6441.0071.51C
ATOM1317OTHRA195−23.636−0.5656.7481.0070.29O
ATOM1318CBTHRA195−25.0342.0925.4031.0073.36C
ATOM1319OG1THRA195−24.3162.4316.5981.0075.79O
ATOM1320CG2THRA195−26.4152.7625.4091.0075.39C
ATOM1321NASNA196−22.8630.0474.7091.0069.76N
ATOM1322CAASNA196−21.483−0.3324.9961.0070.25C
ATOM1323CASNA196−20.8700.8235.7831.0070.56C
ATOM1324OASNA196−21.3501.9675.6941.0073.34O
ATOM1325CBASNA196−20.676−0.7213.7321.0070.71C
ATOM1326CGASNA196−20.4780.4262.7431.0071.43C
ATOM1327OD1ASNA196−19.9401.4813.0711.0074.37O
ATOM1328ND2ASNA196−20.9200.2051.5151.0072.46N
ATOM1329NGLNA197−19.8240.5246.5441.0069.71N
ATOM1330CAGLNA197−19.1951.5077.4461.0069.84C
ATOM1331CGLNA197−18.5702.6876.7111.0068.45C
ATOM1332OGLNA197−18.7453.8327.1371.0068.34O
ATOM1333CBGLNA197−18.1410.8298.3191.0070.49C
ATOM1334CGGLNA197−18.723−0.2219.2641.0073.78C
ATOM1335CDGLNA197−17.662−1.0179.9771.0075.55C
ATOM1336OE1GLNA197−16.556−0.52810.2231.0081.19O
ATOM1337NE2GLNA197−17.988−2.26210.3191.0083.68N
ATOM1338NALAA198−17.8552.4005.6171.0067.03N
ATOM1339CAALAA198−17.2223.4304.7721.0067.06C
ATOM1340CALAA198−18.2294.4804.3091.0068.00C
ATOM1341OALAA198−17.9265.6724.3101.0067.52O
ATOM1342CBALAA198−16.5402.7963.5761.0067.06C
ATOM1343NTYRA199−19.4204.0273.9161.0067.41N
ATOM1344CATYRA199−20.5034.9253.5441.0068.01C
ATOM1345CTYRA199−21.0015.6784.7531.0066.60C
ATOM1346OTYRA199−21.1626.8894.6971.0064.41O
ATOM1347CBTYRA199−21.6854.1682.9351.0069.88C
ATOM1348CGTYRA199−22.8975.0542.7141.0067.04C
ATOM1349CD1TYRA199−23.0095.8311.5661.0070.50C
ATOM1350CD2TYRA199−23.9325.1133.6551.0069.18C
ATOM1351CE1TYRA199−24.1286.6571.3521.0071.51C
ATOM1352CE2TYRA199−25.0515.9333.4531.0072.40C
ATOM1353CZTYRA199−25.1416.7022.2981.0071.80C
ATOM1354OHTYRA199−26.2367.5092.0881.0073.79O
ATOM1355NALAA200−21.2454.9425.8401.0067.80N
ATOM1356CAALAA200−21.7235.5317.1001.0066.98C
ATOM1357CALAA200−20.8406.6977.5331.0065.35C
ATOM1358OALAA200−21.3547.7397.8901.0062.59O
ATOM1359CBALAA200−21.7894.4868.1831.0067.92C
ATOM1360NILEA201−19.5206.5057.4891.0066.75N
ATOM1361CAILEA201−18.5597.5657.8301.0069.39C
ATOM1362CILEA201−18.5148.6806.7851.0070.99C
ATOM1363OILEA201−18.7689.8367.1071.0071.46O
ATOM1364CBILEA201−17.1267.0277.9711.0068.97C
ATOM1365CG1ILEA201−16.9986.1429.2101.0070.83C
ATOM1366CG2ILEA201−16.1428.1818.0641.0068.40C
ATOM1367CD1ILEA201−15.6105.5309.3831.0071.18C
ATOM1368NALAA202−18.1918.3115.5421.0071.53N
ATOM1369CAALAA202−18.0019.2734.4381.0070.39C
ATOM1370CALAA202−19.22810.1124.1361.0071.21C
ATOM1371OALAA202−19.11211.3313.9791.0072.58O
ATOM1372CBALAA202−17.5588.5553.1771.0069.73C
ATOM1373NSERA203−20.3959.4744.0541.0071.88N
ATOM1374CASERA203−21.63310.2023.7481.0072.73C
ATOM1375CSERA203−22.03011.1604.8611.0075.38C
ATOM1376OSERA203−22.45712.2744.5791.0076.69O
ATOM1377CBSERA203−22.7989.2613.4921.0073.42C
ATOM1378OGSERA203−23.94410.0103.1181.0077.91O
ATOM1379NSERA204−21.89010.7306.1141.0072.53N
ATOM1380CASERA204−22.26611.5707.2501.0071.77C
ATOM1381CSERA204−21.28312.7427.4421.0073.36C
ATOM1382OSERA204−21.69513.8227.8631.0073.43O
ATOM1383CBSERA204−22.42810.7288.5251.0069.52C
ATOM1384OGSERA204−21.2929.9418.7761.0076.74O
ATOM1385NILEA205−19.99812.5377.1371.0073.22N
ATOM1386CAILEA205−19.02013.6387.2131.0073.31C
ATOM1387CILEA205−19.28414.6746.1181.0076.06C
ATOM1388OILEA205−19.39915.8726.4051.0076.12O
ATOM1389CBILEA205−17.56913.1477.1261.0072.39C
ATOM1390CG1ILEA205−17.17312.4588.4351.0071.69C
ATOM1391CG2ILEA205−16.62514.3036.8761.0070.67C
ATOM1392CD1ILEA205−15.81211.7948.3901.0072.10C
ATOM1393NVALA206−19.38014.2014.8771.0074.65N
ATOM1394CAVALA206−19.59115.0813.7131.0077.11C
ATOM1395CVALA206−20.97215.7573.6651.0075.89C
ATOM1396OVALA206−21.07116.9333.3171.0075.71O
ATOM1397CBVALA206−19.35614.3212.3881.0075.98C
ATOM1398CG1VALA206−19.64715.2101.1941.0077.29C
ATOM1399CG2VALA206−17.92013.8192.3241.0080.65C
ATOM1400NSERA207−22.02315.0254.0091.0076.66N
ATOM1401CASERA207−23.37715.5853.9721.0077.79C
ATOM1402CSERA207−23.72016.4575.1651.0078.97C
ATOM1403OSERA207−24.43717.4374.9951.0083.06O
ATOM1404CBSERA207−24.42014.4883.9221.0078.06C
ATOM1405OGSERA207−24.23113.6332.8141.0083.89O
ATOM1406NPHEA208−23.21616.1036.3561.0079.98N
ATOM1407CAPHEA208−23.60116.7717.6091.0077.07C
ATOM1408CPHEA208−22.46317.4778.3191.0078.35C
ATOM1409OPHEA208−22.51118.6958.4701.0079.76O
ATOM1410CBPHEA208−24.25415.7538.5521.0076.98C
ATOM1411CGPHEA208−24.67616.3219.8881.0078.18C
ATOM1412CD1PHEA208−25.79217.1419.9841.0077.60C
ATOM1413CD2PHEA208−23.95716.02511.0511.0075.04C
ATOM1414CE1PHEA208−26.19017.66811.2161.0077.26C
ATOM1415CE2PHEA208−24.35016.54512.2781.0080.26C
ATOM1416CZPHEA208−25.47517.37312.3571.0077.27C
ATOM1417NTYRA209−21.45016.7398.7561.0078.29N
ATOM1418CATYRA209−20.38617.3519.5931.0078.96C
ATOM1419CTYRA209−19.58318.4638.9411.0078.89C
ATOM1420OTYRA209−19.22219.4229.6251.0079.13O
ATOM1421CBTYRA209−19.42516.29510.1311.0080.47C
ATOM1422CGTYRA209−20.04715.42511.1811.0082.05C
ATOM1423CD1TYRA209−20.33014.08810.9371.0080.35C
ATOM1424CD2TYRA209−20.36015.94812.4401.0084.46C
ATOM1425CE1TYRA209−20.90613.29211.9131.0083.44C
ATOM1426CE2TYRA209−20.93615.16313.4241.0085.84C
ATOM1427CZTYRA209−21.20813.83513.1591.0085.25C
ATOM1428OHTYRA209−21.77613.07314.1451.0089.70O
ATOM1429NVALA210−19.30418.3517.6451.0078.18N
ATOM1430CAVALA210−18.55719.4046.9441.0077.41C
ATOM1431CVALA210−19.35020.7406.9441.0077.38C
ATOM1432OVALA210−18.85321.7167.4951.0080.02O
ATOM1433CBVALA210−18.10818.9765.5151.0078.51C
ATOM1434CG1VALA210−17.75820.2024.6531.0073.94C
ATOM1435CG2VALA210−16.93218.0225.6091.0076.43C
ATOM1436NPROA211−20.55720.7806.3391.0075.78N
ATOM1437CAPROA211−21.30422.0336.3791.0076.70C
ATOM1438CPROA211−21.72122.5097.7931.0078.77C
ATOM1439OPROA211−21.91723.7077.9671.0081.95O
ATOM1440CBPROA211−22.53821.7385.5061.0078.00C
ATOM1441CGPROA211−22.68720.3075.5141.0077.13C
ATOM1442CDPROA211−21.30519.7465.6011.0080.23C
ATOM1443NLEUA212−21.84821.5988.7671.0077.02N
ATOM1444CALEUA212−22.17821.96510.1591.0075.25C
ATOM1445CLEUA212−21.03422.72310.8151.0075.40C
ATOM1446OLEUA212−21.24823.76511.4041.0076.71O
ATOM1447CBLEUA212−22.49220.72711.0171.0073.79C
ATOM1448CGLEUA212−22.75920.90612.5371.0077.60C
ATOM1449CD1LEUA212−24.10221.57112.7591.0079.25C
ATOM1450CD2LEUA212−22.69119.55813.3061.0070.72C
ATOM1451NVALA213−19.82622.18310.7111.0076.14N
ATOM1452CAVALA213−18.63622.80811.3111.0076.70C
ATOM1453CVALA213−18.37224.19010.7131.0077.08C
ATOM1454OVALA213−18.03925.13211.4371.0076.69O
ATOM1455CBVALA213−17.38021.91511.1461.0079.00C
ATOM1456CG1VALA213−16.11122.65611.5621.0078.68C
ATOM1457CG2VALA213−17.53420.62711.9551.0077.70C
ATOM1458NILEA214−18.52824.2959.3961.0074.65N
ATOM1459CAILEA214−18.37225.5618.6901.0074.83C
ATOM1460CILEA214−19.42326.5749.1621.0072.99C
ATOM1461OILEA214−19.07527.6929.5021.0072.81O
ATOM1462CBILEA214−18.48025.3847.1441.0073.17C
ATOM1463CG1ILEA214−17.31824.5476.6031.0076.80C
ATOM1464CG2ILEA214−18.50126.7486.4501.0071.35C
ATOM1465CD1ILEA214−17.44324.2025.1261.0072.96C
ATOM1466NMETA215−20.69226.1599.1711.0073.55N
ATOM1467CAMETA215−21.82426.9909.6111.0075.44C
ATOM1468CMETA215−21.58927.58010.9791.0077.02C
ATOM1469OMETA215−21.69028.78711.1501.0079.77O
ATOM1470CBMETA215−23.09926.1539.6751.0074.74C
ATOM1471CGMETA215−24.37426.87410.1241.0073.35C
ATOM1472SDMETA215−25.65325.67210.5451.0082.79S
ATOM1473CEMETA215−25.10225.18512.1881.0078.98C
ATOM1474NVALA216−21.28126.70911.9381.0073.57N
ATOM1475CAVALA216−21.02927.11113.3191.0077.98C
ATOM1476CVALA216−19.84728.07713.4241.0078.25C
ATOM1477OVALA216−19.96029.12414.0611.0082.48O
ATOM1478CBVALA216−20.76025.88814.2491.0074.95C
ATOM1479CG1VALA216−20.35826.35015.6131.0073.89C
ATOM1480CG2VALA216−21.99524.99214.3341.0072.42C
ATOM1481NPHEA217−18.72427.72412.8021.0078.36N
ATOM1482CAPHEA217−17.55928.59512.8201.0077.83C
ATOM1483CPHEA217−17.88029.96212.2271.0074.98C
ATOM1484OPHEA217−17.66130.98112.8621.0074.40O
ATOM1485CBPHEA217−16.39427.99212.0541.0080.26C
ATOM1486CGPHEA217−15.23628.93011.9181.0083.15C
ATOM1487CD1PHEA217−14.38129.15212.9941.0087.33C
ATOM1488CD2PHEA217−14.99329.60010.7171.0087.93C
ATOM1489CE1PHEA217−13.29630.02912.8811.0085.31C
ATOM1490CE2PHEA217−13.91230.47810.5921.0087.26C
ATOM1491CZPHEA217−13.06330.69211.6771.0086.57C
ATOM1492NVALA218−18.40029.95811.0091.0072.95N
ATOM1493CAVALA218−18.69331.20210.2991.0072.35C
ATOM1494CVALA218−19.73932.03911.0071.0070.18C
ATOM1495OVALA218−19.54733.23811.1231.0072.08O
ATOM1496CBVALA218−19.12630.9658.8281.0069.97C
ATOM1497CG1VALA218−19.60832.2648.1891.0063.51C
ATOM1498CG2VALA218−17.97330.3938.0391.0069.43C
ATOM1499NTYRA219−20.82231.42811.4791.0069.91N
ATOM1500CATYRA219−21.86732.20412.1541.0070.79C
ATOM1501CTYRA219−21.48232.64613.5611.0072.41C
ATOM1502OTYRA219−22.13533.53214.0941.0073.85O
ATOM1503CBTYRA219−23.21631.48212.2011.0070.68C
ATOM1504CGTYRA219−24.37332.46812.1491.0072.35C
ATOM1505CD1TYRA219−24.69833.11210.9681.0075.81C
ATOM1506CD2TYRA219−25.13632.75513.2631.0077.93C
ATOM1507CE1TYRA219−25.75734.02510.8921.0074.63C
ATOM1508CE2TYRA219−26.20333.67313.1901.0070.80C
ATOM1509CZTYRA219−26.49934.29212.0131.0079.33C
ATOM1510OHTYRA219−27.54435.18711.9581.0075.98O
ATOM1511NSERA220−20.45132.05314.1701.0071.26N
ATOM1512CASERA220−19.98232.57915.4561.0072.46C
ATOM1513CSERA220−19.26533.88615.1241.0073.33C
ATOM1514OSERA220−19.38434.83315.8761.0071.17O
ATOM1515CBSERA220−19.11031.59316.2401.0071.03C
ATOM1516OGSERA220−17.98831.16915.5141.0078.76O
ATOM1517NARGA221−18.53933.92413.9901.0071.00N
ATOM1518CAARGA221−17.87835.16413.5071.0071.92C
ATOM1519CARGA221−18.84736.26813.1021.0071.03C
ATOM1520OARGA221−18.48537.44013.1441.0075.74O
ATOM1521CBARGA221−16.94034.88712.3191.0073.28C
ATOM1522CGARGA221−15.78933.96712.6071.0081.79C
ATOM1523CDARGA221−14.73734.64313.4601.0091.32C
ATOM1524NEARGA221−13.69533.71113.9001.0099.40N
ATOM1525CZARGA221−12.64234.03714.6691.00104.74C
ATOM1526NH1ARGA221−12.46335.29615.1071.00108.58N
ATOM1527NH2ARGA221−11.75433.09515.0091.00102.27N
ATOM1528NVALA222−20.06535.89812.7051.0072.12N
ATOM1529CAVALA222−21.11436.86212.3901.0070.08C
ATOM1530CVALA222−21.54437.55513.6801.0072.52C
ATOM1531OVALA222−21.66638.78713.7041.0074.66O
ATOM1532CBVALA222−22.31436.19911.6881.0069.66C
ATOM1533CG1VALA222−23.52737.09911.7131.0070.73C
ATOM1534CG2VALA222−21.94035.84410.2451.0074.61C
ATOM1535NPHEA223−21.77136.78214.7521.0072.29N
ATOM1536CAPHEA223−22.11537.38816.0541.0071.42C
ATOM1537CPHEA223−21.02038.30316.5621.0069.83C
ATOM1538OPHEA223−21.32439.41116.9701.0071.03O
ATOM1539CBPHEA223−22.46136.33417.1051.0072.71C
ATOM1540CGPHEA223−23.76635.64016.8571.0069.91C
ATOM1541CD1PHEA223−24.92736.37016.5631.0071.96C
ATOM1542CD2PHEA223−23.85034.26616.9201.0073.93C
ATOM1543CE1PHEA223−26.12135.74116.3381.0073.93C
ATOM1544CE2PHEA223−25.05533.62416.6931.0076.95C
ATOM1545CZPHEA223−26.19034.36716.4041.0078.80C
ATOM1546NGLNA224−19.76637.85416.5341.0070.23N
ATOM1547CAGLNA224−18.63238.72416.9161.0071.28C
ATOM1548CGLNA224−18.65540.03416.1551.0072.34C
ATOM1549OGLNA224−18.46241.07716.7421.0076.18O
ATOM1550CBGLNA224−17.29338.07716.6391.0071.04C
ATOM1551CGGLNA224−17.00736.80717.4331.0082.15C
ATOM1552CDGLNA224−15.56536.30817.2761.0085.53C
ATOM1553OE1GLNA224−14.70636.98616.6731.0085.57O
ATOM1554NE2GLNA224−15.29035.10717.8241.0087.83N
ATOM1555NGLUA225−18.89239.96914.8391.0074.23N
ATOM1556CAGLUA225−18.98641.18714.0261.0073.90C
ATOM1557CGLUA225−20.16142.04314.4421.0072.00C
ATOM1558OGLUA225−19.98743.22114.6701.0069.39O
ATOM1559CBGLUA225−19.07340.88012.5251.0075.78C
ATOM1560CGGLUA225−17.71340.80611.8811.0085.48C
ATOM1561CDGLUA225−16.99042.13411.9341.0092.43C
ATOM1562OE1GLUA225−17.67043.17411.7591.0086.05O
ATOM1563OE2GLUA225−15.75542.13312.1521.0098.53O
ATOM1564NALAA226−21.33641.43914.5371.0070.43N
ATOM1565CAALAA226−22.52142.14514.9881.0071.14C
ATOM1566CALAA226−22.23842.86116.3261.0070.13C
ATOM1567OALAA226−22.51344.04016.4551.0069.11O
ATOM1568CBALAA226−23.68341.19515.1141.0069.59C
ATOM1569NLYSA227−21.68642.13917.2981.0070.92N
ATOM1570CALYSA227−21.34542.71618.6191.0072.35C
ATOM1571CLYSA227−20.37443.88018.5101.0070.68C
ATOM1572OLYSA227−20.49844.86019.2381.0071.56O
ATOM1573CBLYSA227−20.72841.66019.5401.0072.29C
ATOM1574CGLYSA227−20.67342.07721.0271.0077.11C
ATOM1575CDLYSA227−20.27240.91321.9481.0079.33C
ATOM1576CELYSA227−20.36641.29523.4571.0089.68C
ATOM1577NZLYSA227−19.21142.08423.9751.0091.63N
ATOM1578NARGA228−19.41943.74917.5911.0071.06N
ATOM1579CAARGA228−18.38044.74917.3491.0071.67C
ATOM1580CARGA228−18.90946.02816.6961.0066.45C
ATOM1581OARGA228−18.23447.03116.7141.0070.70O
ATOM1582CBARGA228−17.27144.14216.4861.0073.73C
ATOM1583CGARGA228−15.95944.94616.3951.0080.33C
ATOM1584CDARGA228−15.06044.48715.2391.0085.20C
ATOM1585NEARGA228−15.69144.69913.9311.0095.73N
ATOM1586CZARGA228−15.73645.85313.2391.0099.68C
ATOM1587NH1ARGA228−15.17846.97913.7021.0098.15N
ATOM1588NH2ARGA228−16.35345.88912.0521.0097.65N
ATOM1589NGLNA229−20.10546.00216.1271.0067.44N
ATOM1590CAGLNA229−20.71847.21715.5621.0069.07C
ATOM1591CGLNA229−21.32448.10716.6301.0068.99C
ATOM1592OGLNA229−21.55049.29916.3881.0073.07O
ATOM1593CBGLNA229−21.81746.85814.6031.0067.27C
ATOM1594CGGLNA229−21.32846.08513.4031.0079.11C
ATOM1595CDGLNA229−22.41245.83612.3581.0081.84C
ATOM1596OE1GLNA229−22.09745.43711.2331.0098.22O
ATOM1597NE2GLNA229−23.70346.06912.7241.0080.39N
ATOM1598NLEUA230−21.59447.54617.8041.0065.76N
ATOM1599CALEUA230−22.18048.31818.8871.0066.37C
ATOM1600CLEUA230−21.22849.42419.2681.0062.49C
ATOM1601OLEUA230−20.06849.18419.5171.0063.24O
ATOM1602CBLEUA230−22.49247.42920.0781.0063.29C
ATOM1603CGLEUA230−23.52646.34619.8051.0063.55C
ATOM1604CD1LEUA230−23.65945.40121.0171.0056.14C
ATOM1605CD2LEUA230−24.86746.99819.3981.0063.01C
ATOM1606NASNA1002−21.74550.64519.2981.0065.96N
ATOM1607CAASNA1002−20.95951.84619.6321.0068.64C
ATOM1608CASNA1002−21.91152.94620.0591.0068.56C
ATOM1609OASNA1002−23.12552.70620.1341.0070.46O
ATOM1610CBASNA1002−20.11452.26018.4421.0068.39C
ATOM1611CGASNA1002−20.93552.51717.2241.0074.98C
ATOM1612OD1ASNA1002−22.10052.93417.3131.0065.74O
ATOM1613ND2ASNA1002−20.34252.27316.0581.0067.18N
ATOM1614NILEA1003−21.39054.13920.3301.0069.49N
ATOM1615CAILEA1003−22.22455.20320.9011.0070.87C
ATOM1616CILEA1003−23.40455.57220.0131.0070.78C
ATOM1617OILEA1003−24.47255.88620.5211.0074.04O
ATOM1618CBILEA1003−21.36256.45321.3101.0074.80C
ATOM1619CG1ILEA1003−22.17257.39022.2291.0076.63C
ATOM1620CG2ILEA1003−20.78857.15320.0751.0068.47C
ATOM1621CD1ILEA1003−21.37058.56022.7371.0073.42C
ATOM1622NPHEA1004−23.20455.52718.6951.0073.63N
ATOM1623CAPHEA1004−24.26155.80117.7191.0072.72C
ATOM1624CPHEA1004−25.40954.84117.8351.0073.43C
ATOM1625OPHEA1004−26.57455.25517.8721.0071.77O
ATOM1626CBPHEA1004−23.67955.72716.3091.0075.62C
ATOM1627CGPHEA1004−24.68955.71615.2361.0071.84C
ATOM1628CD1PHEA1004−25.45356.84814.9791.0082.28C
ATOM1629CD2PHEA1004−24.89254.57014.4521.0082.90C
ATOM1630CE1PHEA1004−26.42356.84313.9431.0077.44C
ATOM1631CE2PHEA1004−25.86554.56413.4101.0071.41C
ATOM1632CZPHEA1004−26.62055.69713.1661.0070.16C
ATOM1633NGLUA1005−25.07453.55917.8891.0072.98N
ATOM1634CAGLUA1005−26.06352.51718.0641.0072.41C
ATOM1635CGLUA1005−26.73352.61919.4161.0074.97C
ATOM1636OGLUA1005−27.94952.43419.5171.0074.19O
ATOM1637CBGLUA1005−25.41851.13017.9311.0072.97C
ATOM1638CGGLUA1005−24.75950.84416.5611.0075.62C
ATOM1639CDGLUA1005−25.71351.00815.3961.0084.27C
ATOM1640OE1GLUA1005−26.92550.89915.6401.0091.81O
ATOM1641OE2GLUA1005−25.26151.24314.2471.0080.28O
ATOM1642NMETA1006−25.93952.91320.4531.0071.72N
ATOM1643CAMETA1006−26.46753.10121.7831.0072.95C
ATOM1644CMETA1006−27.56254.18221.7961.0073.35C
ATOM1645OMETA1006−28.63953.96322.3561.0073.69O
ATOM1646CBMETA1006−25.31553.43822.7731.0073.49C
ATOM1647CGMETA1006−25.76953.93324.1311.0078.05C
ATOM1648SDMETA1006−24.44754.46825.2541.0074.91S
ATOM1649CEMETA1006−24.29353.08326.3961.0086.03C
ATOM1650NLEUA1007−27.28655.33521.1791.0072.95N
ATOM1651CALEUA1007−28.23556.46421.2091.0073.25C
ATOM1652CLEUA1007−29.34756.26820.1931.0074.44C
ATOM1653OLEUA1007−30.44156.79520.3521.0075.83O
ATOM1654CBLEUA1007−27.53957.81421.0031.0071.83C
ATOM1655CGLEUA1007−26.95358.59322.1971.0076.11C
ATOM1656CD1LEUA1007−28.03158.91623.2481.0080.06C
ATOM1657CD2LEUA1007−25.78657.88622.8291.0073.36C
ATOM1658NARGA1008−29.06955.51419.1491.0077.47N
ATOM1659CAARGA1008−30.09555.13718.1831.0077.36C
ATOM1660CARGA1008−31.13854.23118.8651.0076.88C
ATOM1661OARGA1008−32.31154.32918.5751.0075.51O
ATOM1662CBARGA1008−29.43754.43017.0401.0072.89C
ATOM1663CGARGA1008−30.32554.07715.9361.0083.81C
ATOM1664CDARGA1008−29.53053.46014.8401.0080.34C
ATOM1665NEARGA1008−30.36852.53414.0931.0081.78N
ATOM1666CZARGA1008−29.94451.43113.4851.0085.98C
ATOM1667NH1ARGA1008−28.66051.08113.5241.0080.09N
ATOM1668NH2ARGA1008−30.82350.66012.8231.0093.61N
ATOM1669NILEA1009−30.67853.35519.7731.0078.10N
ATOM1670CAILEA1009−31.54852.46520.5431.0075.58C
ATOM1671CILEA1009−32.25153.25121.6581.0078.69C
ATOM1672OILEA1009−33.45553.13421.8261.0081.42O
ATOM1673CBILEA1009−30.73651.27421.1551.0078.60C
ATOM1674CG1ILEA1009−30.22050.32920.0571.0070.72C
ATOM1675CG2ILEA1009−31.56850.49322.2021.0073.84C
ATOM1676CD1ILEA1009−28.92449.57920.4471.0068.79C
ATOM1677NASPA1010−31.49254.04922.4121.0081.55N
ATOM1678CAASPA1010−32.04854.81323.5551.0080.99C
ATOM1679CASPA1010−32.89256.04523.1991.0082.41C
ATOM1680OASPA1010−33.89256.30223.8761.0083.53O
ATOM1681CBASPA1010−30.91855.25424.4861.0079.05C
ATOM1682CGASPA1010−30.27354.10325.2161.0081.30C
ATOM1683OD1ASPA1010−30.91853.07625.3541.0079.79O
ATOM1684OD2ASPA1010−29.11054.21725.6601.0068.53O
ATOM1685NGLUA1011−32.49956.79022.1631.0081.15N
ATOM1686CAGLUA1011−33.20658.04621.7491.0082.09C
ATOM1687CGLUA1011−34.07557.89820.4991.0082.84C
ATOM1688OGLUA1011−35.12458.54120.3971.0086.08O
ATOM1689CBGLUA1011−32.18959.19721.5481.0085.07C
ATOM1690CGGLUA1011−32.74860.53520.9871.0086.91C
ATOM1691CDGLUA1011−33.83461.16621.8571.0092.06C
ATOM1692OE1GLUA1011−33.79160.98023.0981.0092.05O
ATOM1693OE2GLUA1011−34.73661.85521.2911.0082.39O
ATOM1694NGLYA1012−33.64857.06719.5501.0077.24N
ATOM1695CAGLYA1012−34.40756.83918.3361.0075.19C
ATOM1696CGLYA1012−33.92557.76817.2461.0074.64C
ATOM1697OGLYA1012−33.63558.93417.4941.0071.63O
ATOM1698NLEUA1013−33.84157.24516.0321.0074.33N
ATOM1699CALEUA1013−33.40258.02414.8921.0073.75C
ATOM1700CLEUA1013−34.65358.39414.1031.0073.66C
ATOM1701OLEUA1013−35.39757.51113.6701.0072.82O
ATOM1702CBLEUA1013−32.41957.21414.0601.0073.46C
ATOM1703CGLEUA1013−31.52058.01413.1181.0077.16C
ATOM1704CD1LEUA1013−30.40057.16112.6111.0081.09C
ATOM1705CD2LEUA1013−32.30458.57311.9781.0075.18C
ATOM1706NARGA1014−34.87559.69613.9251.0073.47N
ATOM1707CAARGA1014−36.00560.20613.1431.0075.08C
ATOM1708CARGA1014−35.49260.91511.8941.0073.46C
ATOM1709OARGA1014−34.66561.81811.9671.0072.29O
ATOM1710CBARGA1014−36.89961.09813.9941.0073.94C
ATOM1711CGARGA1014−37.55260.28615.1281.0082.18C
ATOM1712CDARGA1014−38.78360.91715.7161.0087.29C
ATOM1713NEARGA1014−39.87961.08914.7461.0093.86N
ATOM1714CZARGA1014−41.12961.46915.0531.0092.93C
ATOM1715NH1ARGA1014−41.48861.72916.3231.0095.92N
ATOM1716NH2ARGA1014−42.03861.59114.0801.0092.74N
ATOM1717NLEUA1015−36.00160.48110.7461.0073.79N
ATOM1718CALEUA1015−35.56660.9699.4441.0073.57C
ATOM1719CLEUA1015−36.28462.2399.0331.0074.92C
ATOM1720OLEUA1015−35.75762.9888.2241.0072.14O
ATOM1721CBLEUA1015−35.78159.8718.4111.0073.34C
ATOM1722CGLEUA1015−35.06558.5558.7491.0071.48C
ATOM1723CD1LEUA1015−35.24057.5767.6261.0066.34C
ATOM1724CD2LEUA1015−33.59558.7839.0331.0063.66C
ATOM1725NLYSA1016−37.48062.4729.5931.0077.95N
ATOM1726CALYSA1016−38.27463.6789.3271.0075.82C
ATOM1727CLYSA1016−38.33864.57110.5631.0073.96C
ATOM1728OLYSA1016−38.37964.06511.6801.0071.82O
ATOM1729CBLYSA1016−39.68863.3028.9081.0075.21C
ATOM1730CGLYSA1016−39.74462.4517.6491.0079.49C
ATOM1731CDLYSA1016−41.15962.2147.1761.0077.17C
ATOM1732CELYSA1016−41.16061.3585.9001.0081.57C
ATOM1733NZLYSA1016−42.52161.1875.2961.0074.99N
ATOM1734NILEA1017−38.34765.88910.3341.0073.18N
ATOM1735CAILEA1017−38.51166.90011.3721.0072.14C
ATOM1736CILEA1017−39.70366.59512.2651.0073.25C
ATOM1737OILEA1017−40.76466.20711.7741.0074.28O
ATOM1738CBILEA1017−38.70568.32010.7651.0071.53C
ATOM1739CG1ILEA1017−37.39168.86210.2101.0074.05C
ATOM1740CG2ILEA1017−39.20469.30811.7881.0070.34C
ATOM1741CD1ILEA1017−37.44370.3189.8331.0073.51C
ATOM1742NTYRA1018−39.51566.77313.5731.0072.91N
ATOM1743CATYRA1018−40.57166.56214.5521.0074.12C
ATOM1744CTYRA1018−40.39167.54015.7001.0073.99C
ATOM1745OTYRA1018−39.30468.09115.8831.0075.95O
ATOM1746CBTYRA1018−40.54665.11915.0701.0075.31C
ATOM1747CGTYRA1018−39.31664.76915.8841.0075.01C
ATOM1748CD1TYRA1018−39.35564.76517.2831.0077.73C
ATOM1749CD2TYRA1018−38.11364.44015.2631.0075.41C
ATOM1750CE1TYRA1018−38.22364.44218.0421.0072.04C
ATOM1751CE2TYRA1018−36.97464.11616.0161.0077.21C
ATOM1752CZTYRA1018−37.04264.12017.4031.0076.64C
ATOM1753OHTYRA1018−35.92363.80018.1421.0079.46O
ATOM1754NLYSA1019−41.46167.74616.4591.0073.71N
ATOM1755CALYSA1019−41.42768.59717.6361.0074.41C
ATOM1756CLYSA1019−41.05667.72918.8261.0075.37C
ATOM1757OLYSA1019−41.71566.71819.0731.0077.13O
ATOM1758CBLYSA1019−42.77369.26217.8851.0073.28C
ATOM1759CGLYSA1019−43.10470.35216.8991.0072.01C
ATOM1760CDLYSA1019−44.35771.09917.3261.0072.42C
ATOM1761CELYSA1019−44.64972.28016.4141.0073.72C
ATOM1762NZLYSA1019−45.91072.99316.7961.0071.74N
ATOM1763NASPA1020−40.01268.11219.5611.0076.71N
ATOM1764CAASPA1020−39.61067.38820.7761.0078.54C
ATOM1765CASPA1020−40.62967.61521.9131.0078.53C
ATOM1766OASPA1020−41.69368.20521.6981.0079.88O
ATOM1767CBASPA1020−38.17467.77821.1981.0079.44C
ATOM1768CGASPA1020−38.07169.18321.8381.0081.53C
ATOM1769OD1ASPA1020−39.08769.87322.0691.0077.18O
ATOM1770OD2ASPA1020−36.92969.60022.1161.0089.87O
ATOM1771NTHRA1021−40.29767.14723.1121.0081.31N
ATOM1772CATHRA1021−41.16367.30324.2991.0081.40C
ATOM1773CTHRA1021−41.46468.77124.6741.0081.89C
ATOM1774OTHRA1021−42.56269.08525.1521.0083.48O
ATOM1775CBTHRA1021−40.53266.62425.5141.0083.23C
ATOM1776OG1THRA1021−39.21267.14925.7161.0086.46O
ATOM1777CG2THRA1021−40.45465.10025.3051.0083.63C
ATOM1778NGLUA1022−40.48869.65324.4471.0081.25N
ATOM1779CAGLUA1022−40.61771.09124.7201.0080.42C
ATOM1780CGLUA1022−41.25671.86323.5341.0079.28C
ATOM1781OGLUA1022−41.33973.09323.5761.0079.62O
ATOM1782CBGLUA1022−39.24371.71025.0321.0081.04C
ATOM1783CGGLUA1022−38.30170.94026.0111.0082.43C
ATOM1784CDGLUA1022−38.78870.89127.4481.0092.01C
ATOM1785OE1GLUA1022−39.74971.61127.8061.0094.63O
ATOM1786OE2GLUA1022−38.19270.11728.2311.0097.00O
ATOM1787NGLYA1023−41.69671.14622.4911.0079.51N
ATOM1788CAGLYA1023−42.31071.74321.3021.0078.42C
ATOM1789CGLYA1023−41.32472.23220.2441.0078.89C
ATOM1790OGLYA1023−41.75072.84219.2551.0079.69O
ATOM1791NTYRA1024−40.02171.97120.4361.0077.49N
ATOM1792CATYRA1024−38.98172.47619.5321.0076.94C
ATOM1793CTYRA1024−38.69471.54818.3631.0077.71C
ATOM1794OTYRA1024−38.68670.32118.5241.0079.61O
ATOM1795CBTYRA1024−37.66372.69920.2691.0077.27C
ATOM1796CGTYRA1024−37.71173.69221.4001.0079.68C
ATOM1797CD1TYRA1024−38.14574.98721.1921.0081.27C
ATOM1798CD2TYRA1024−37.31373.33722.6841.0079.56C
ATOM1799CE1TYRA1024−38.18975.91322.2321.0083.68C
ATOM1800CE2TYRA1024−37.35374.25323.7361.0078.83C
ATOM1801CZTYRA1024−37.79275.54123.5031.0080.59C
ATOM1802OHTYRA1024−37.83676.45724.5341.0083.58O
ATOM1803NTYRA1025−38.45272.14517.1911.0074.53N
ATOM1804CATYRA1025−38.17671.38715.9691.0073.53C
ATOM1805CTYRA1025−36.84970.65716.0501.0068.58C
ATOM1806OTYRA1025−35.80171.28516.1881.0069.06O
ATOM1807CBTYRA1025−38.19572.29514.7361.0071.74C
ATOM1808CGTYRA1025−39.56472.80614.4161.0070.34C
ATOM1809CD1TYRA1025−39.92474.10414.7171.0071.87C
ATOM1810CD2TYRA1025−40.51071.98313.8101.0071.28C
ATOM1811CE1TYRA1025−41.18674.57714.4231.0077.57C
ATOM1812CE2TYRA1025−41.78372.44513.5091.0070.46C
ATOM1813CZTYRA1025−42.11873.74713.8191.0072.50C
ATOM1814OHTYRA1025−43.36674.22213.5311.0073.02O
ATOM1815NTHRA1026−36.92369.33015.9601.0068.83N
ATOM1816CATHRA1026−35.78368.43116.1291.0069.83C
ATOM1817CTHRA1026−35.73067.40815.0001.0071.01C
ATOM1818OTHRA1026−36.75267.15314.3381.0075.69O
ATOM1819CBTHRA1026−35.92267.74117.5001.0068.73C
ATOM1820OG1THRA1026−36.03368.75218.5021.0071.36O
ATOM1821CG2THRA1026−34.75066.82417.8431.0065.86C
ATOM1822NILEA1027−34.55266.82514.7691.0069.80N
ATOM1823CAILEA1027−34.39765.78813.7371.0072.31C
ATOM1824CILEA1027−33.25964.83614.0911.0073.35C
ATOM1825OILEA1027−32.33965.21514.7941.0074.44O
ATOM1826CBILEA1027−34.15366.42812.3501.0071.44C
ATOM1827CG1ILEA1027−34.48265.44311.2241.0073.60C
ATOM1828CG2ILEA1027−32.71366.93412.2351.0069.68C
ATOM1829CD1ILEA1027−34.54066.1009.8551.0069.11C
ATOM1830NGLYA1028−33.33163.59613.6011.0072.50N
ATOM1831CAGLYA1028−32.28762.59913.8631.0071.73C
ATOM1832CGLYA1028−32.36662.10715.2921.0071.31C
ATOM1833OGLYA1028−33.45561.89115.8071.0070.02O
ATOM1834NILEA1029−31.20261.93515.9181.0073.01N
ATOM1835CAILEA1029−31.09061.46017.2951.0072.52C
ATOM1836CILEA1029−31.16762.68718.2321.0074.59C
ATOM1837OILEA1029−30.15863.15218.7821.0077.27O
ATOM1838CBILEA1029−29.78660.63717.5261.0072.24C
ATOM1839CG1ILEA1029−29.69659.44616.5691.0071.48C
ATOM1840CG2ILEA1029−29.71660.14018.9981.0070.08C
ATOM1841CD1ILEA1029−28.29058.89816.3651.0072.58C
ATOM1842NGLYA1030−32.37863.19518.3971.0072.49N
ATOM1843CAGLYA1030−32.62264.37519.1971.0072.04C
ATOM1844CGLYA1030−31.76065.58218.8611.0073.16C
ATOM1845OGLYA1030−31.34966.29119.7551.0072.16O
ATOM1846NHISA1031−31.48065.82117.5741.0075.22N
ATOM1847CAHISA1031−30.70567.00717.1921.0074.36C
ATOM1848CHISA1031−31.66868.17517.1341.0075.22C
ATOM1849OHISA1031−32.61468.13716.3711.0078.88O
ATOM1850CBHISA1031−29.98566.86415.8401.0076.09C
ATOM1851CGHISA1031−29.19068.06815.4701.0074.73C
ATOM1852ND1HISA1031−27.82868.14015.6441.0073.37N
ATOM1853CD2HISA1031−29.56869.25514.9411.0070.52C
ATOM1854CE1HISA1031−27.39769.32015.2341.0072.05C
ATOM1855NE2HISA1031−28.43470.01614.8051.0073.60N
ATOM1856NLEUA1032−31.42069.20917.9371.0074.86N
ATOM1857CALEUA1032−32.27170.40017.9661.0075.57C
ATOM1858CLEUA1032−31.87171.33416.8401.0075.37C
ATOM1859OLEUA1032−30.68171.65416.6901.0076.52O
ATOM1860CBLEUA1032−32.14871.13719.3051.0074.42C
ATOM1861CGLEUA1032−33.09272.32619.5451.0076.35C
ATOM1862CD1LEUA1032−34.49071.82119.7221.0081.83C
ATOM1863CD2LEUA1032−32.68273.12420.7601.0073.47C
ATOM1864NLEUA1033−32.85771.77016.0571.0074.07N
ATOM1865CALEUA1033−32.61072.67514.9281.0073.31C
ATOM1866CLEUA1033−32.68474.14715.3311.0072.89C
ATOM1867OLEUA1033−31.78974.93715.0031.0070.82O
ATOM1868CBLEUA1033−33.59572.37413.7871.0073.71C
ATOM1869CGLEUA1033−33.38071.03213.0991.0068.93C
ATOM1870CD1LEUA1033−34.52770.72712.1461.0071.95C
ATOM1871CD2LEUA1033−32.02371.02512.3721.0066.34C
ATOM1872NTHRA1034−33.75174.50216.0391.0073.89N
ATOM1873CATHRA1034−33.98675.87316.4941.0071.83C
ATOM1874CTHRA1034−35.07075.84917.5381.0073.47C
ATOM1875OTHRA1034−35.84974.89617.5911.0074.91O
ATOM1876CBTHRA1034−34.43976.79215.3251.0071.67C
ATOM1877OG1THRA1034−34.73178.10815.8071.0067.14O
ATOM1878CG2THRA1034−35.68576.23114.6341.0067.88C
ATOM1879NLYSA1035−35.12076.89218.3651.0073.16N
ATOM1880CALYSA1035−36.19077.03719.3521.0074.56C
ATOM1881CLYSA1035−37.40277.82418.7861.0074.05C
ATOM1882OLYSA1035−38.43577.95519.4561.0073.29O
ATOM1883CBLYSA1035−35.66977.66620.6561.0075.36C
ATOM1884CGLYSA1035−34.71176.75121.4411.0074.77C
ATOM1885CDLYSA1035−34.45577.23622.8661.0077.28C
ATOM1886CELYSA1035−33.83678.64122.9241.0082.03C
ATOM1887NZLYSA1035−33.48479.06024.3311.0081.98N
ATOM1888NSERA1036−37.25678.33017.5591.0073.73N
ATOM1889CASERA1036−38.29679.08916.8691.0073.96C
ATOM1890CSERA1036−39.54078.24816.5531.0073.14C
ATOM1891OSERA1036−39.42077.05516.3311.0075.32O
ATOM1892CBSERA1036−37.71379.64915.5611.0074.91C
ATOM1893OGSERA1036−38.68880.26014.7431.0072.52O
ATOM1894NPROA1037−40.74078.87416.5321.0073.94N
ATOM1895CAPROA1037−41.96878.16916.1531.0073.36C
ATOM1896CPROA1037−42.15778.06114.6321.0074.22C
ATOM1897OPROA1037−43.07277.36514.1841.0076.00O
ATOM1898CBPROA1037−43.05479.05816.7321.0072.94C
ATOM1899CGPROA1037−42.49780.41516.5881.0073.18C
ATOM1900CDPROA1037−41.03280.28416.8611.0073.91C
ATOM1901NSERA1038−41.30878.74413.8551.0073.88N
ATOM1902CASERA1038−41.34678.68812.4061.0073.57C
ATOM1903CSERA1038−40.70977.39911.9341.0074.14C
ATOM1904OSERA1038−39.55277.12212.2711.0073.16O
ATOM1905CBSERA1038−40.59479.87211.7971.0073.20C
ATOM1906OGSERA1038−40.48679.73910.3911.0072.62O
ATOM1907NLEUA1039−41.46376.61711.1631.0072.03N
ATOM1908CALEUA1039−40.96175.37810.5901.0072.88C
ATOM1909CLEUA1039−39.90575.7049.5571.0072.75C
ATOM1910OLEUA1039−38.83675.1009.5431.0074.58O
ATOM1911CBLEUA1039−42.09274.5819.9441.0072.16C
ATOM1912CGLEUA1039−41.71773.2679.2431.0071.92C
ATOM1913CD1LEUA1039−41.05372.29910.1981.0068.87C
ATOM1914CD2LEUA1039−42.95172.6278.6011.0071.51C
ATOM1915NASNA1040−40.21876.6658.6941.0074.45N
ATOM1916CAASNA1040−39.28277.1487.6651.0074.48C
ATOM1917CASNA1040−37.91877.5898.2461.0074.80C
ATOM1918OASNA1040−36.89977.4997.5611.0076.00O
ATOM1919CBASNA1040−39.93778.2776.8381.0074.13C
ATOM1920CGASNA1040−41.13577.7795.9851.0073.14C
ATOM1921OD1ASNA1040−41.18376.6195.5551.0070.14O
ATOM1922ND2ASNA1040−42.09578.6655.7481.0066.32N
ATOM1923NALAA1041−37.91878.0589.5001.0074.86N
ATOM1924CAALAA1041−36.68978.40210.2281.0075.02C
ATOM1925CALAA1041−35.88677.13910.6041.0075.73C
ATOM1926OALAA1041−34.66477.15510.5551.0075.98O
ATOM1927CBALAA1041−37.01179.22011.4661.0073.60C
ATOM1928NALAA1042−36.58376.06110.9751.0075.31N
ATOM1929CAALAA1042−35.95374.76211.2361.0074.13C
ATOM1930CALAA1042−35.32974.2129.9561.0075.51C
ATOM1931OALAA1042−34.15273.8449.9461.0075.22O
ATOM1932CBALAA1042−36.96973.78211.7791.0073.73C
ATOM1933NLYSA1043−36.13574.1648.8871.0075.74N
ATOM1934CALYSA1043−35.68973.7287.5471.0076.54C
ATOM1935CLYSA1043−34.44574.4817.0671.0076.74C
ATOM1936OLYSA1043−33.52673.8696.5301.0078.09O
ATOM1937CBLYSA1043−36.82373.8936.5271.0075.43C
ATOM1938CGLYSA1043−37.94672.8796.6971.0076.23C
ATOM1939CDLYSA1043−39.13673.1995.8061.0082.58C
ATOM1940CELYSA1043−40.09472.0105.6711.0085.26C
ATOM1941NZLYSA1043−40.46571.4206.9801.0090.37N
ATOM1942NSERA1044−34.43075.7987.2671.0075.27N
ATOM1943CASERA1044−33.27276.6266.9311.0075.10C
ATOM1944CSERA1044−32.02376.1757.6941.0075.62C
ATOM1945OSERA1044−30.95776.0237.0971.0077.80O
ATOM1946CBSERA1044−33.55778.0967.2321.0075.53C
ATOM1947OGSERA1044−32.46578.9106.8481.0075.67O
ATOM1948NGLUA1045−32.16675.9669.0061.0073.82N
ATOM1949CAGLUA1045−31.06275.4719.8361.0073.20C
ATOM1950CGLUA1045−30.64174.0649.4561.0071.02C
ATOM1951OGLUA1045−29.44873.7529.4941.0071.07O
ATOM1952CBGLUA1045−31.42775.49411.3311.0071.62C
ATOM1953CGGLUA1045−31.65976.88411.9231.0073.11C
ATOM1954CDGLUA1045−30.43177.76211.9491.0076.79C
ATOM1955OE1GLUA1045−29.30877.26011.7471.0080.04O
ATOM1956OE2GLUA1045−30.59578.98012.1771.0085.26O
ATOM1957NLEUA1046−31.61573.2249.1001.0070.49N
ATOM1958CALEUA1046−31.34271.8398.7101.0073.29C
ATOM1959CLEUA1046−30.49271.8077.4551.0073.52C
ATOM1960OLEUA1046−29.45371.1447.4341.0074.73O
ATOM1961CBLEUA1046−32.64471.0648.4691.0075.19C
ATOM1962CGLEUA1046−32.55669.5628.1301.0073.72C
ATOM1963CD1LEUA1046−31.88168.7759.2431.0074.36C
ATOM1964CD2LEUA1046−33.94269.0007.8511.0073.43C
ATOM1965NASPA1047−30.94572.5296.4261.0072.94N
ATOM1966CAASPA1047−30.22572.6455.1481.0075.39C
ATOM1967CASPA1047−28.81273.1615.3431.0074.85C
ATOM1968OASPA1047−27.87572.6344.7581.0076.38O
ATOM1969CBASPA1047−30.95373.5954.1601.0074.34C
ATOM1970CGASPA1047−32.32773.0763.7131.0075.72C
ATOM1971OD1ASPA1047−32.70771.9384.0721.0072.63O
ATOM1972OD2ASPA1047−33.02973.8242.9951.0080.04O
ATOM1973NLYSA1048−28.67774.1936.1681.0075.86N
ATOM1974CALYSA1048−27.37974.7796.4941.0075.59C
ATOM1975CLYSA1048−26.46473.7797.2481.0075.44C
ATOM1976OLYSA1048−25.24173.8107.0881.0075.09O
ATOM1977CBLYSA1048−27.60376.0617.2961.0075.61C
ATOM1978CGLYSA1048−26.36876.8967.5551.0077.43C
ATOM1979CDLYSA1048−26.72978.3777.8381.0078.34C
ATOM1980CELYSA1048−27.67178.5679.0481.0080.49C
ATOM1981NZLYSA1048−27.07478.07310.3221.0079.34N
ATOM1982NALAA1049−27.07372.9038.0551.0073.58N
ATOM1983CAALAA1049−26.34571.8708.8041.0074.82C
ATOM1984CALAA1049−25.95070.6997.9091.0075.13C
ATOM1985OALAA1049−24.79770.2717.9131.0075.27O
ATOM1986CBALAA1049−27.18871.3649.9731.0073.33C
ATOM1987NILEA1050−26.92470.1947.1521.0073.31N
ATOM1988CAILEA1050−26.74669.0386.2551.0073.24C
ATOM1989CILEA1050−25.99469.3784.9511.0072.49C
ATOM1990OILEA1050−25.15568.6024.5001.0072.34O
ATOM1991CBILEA1050−28.12468.3975.9311.0073.95C
ATOM1992CG1ILEA1050−28.75867.8287.2071.0072.34C
ATOM1993CG2ILEA1050−27.99667.2974.9071.0070.31C
ATOM1994CD1ILEA1050−27.96866.6507.8191.0075.79C
ATOM1995NGLYA1051−26.30470.5304.3621.0070.45N
ATOM1996CAGLYA1051−25.62871.0223.1521.0069.29C
ATOM1997CGLYA1051−26.37170.8101.8411.0068.84C
ATOM1998OGLYA1051−25.74170.6240.8001.0064.99O
ATOM1999NARGA1052−27.70370.8411.9001.0066.56N
ATOM2000CAARGA1052−28.56370.6800.7301.0067.90C
ATOM2001CARGA1052−29.99571.0121.0901.0068.30C
ATOM2002OARGA1052−30.30971.1752.2611.0071.80O
ATOM2003CBARGA1052−28.50269.2510.1621.0066.98C
ATOM2004CGARGA1052−28.97168.1481.0761.0072.44C
ATOM2005CDARGA1052−29.03466.8330.3301.0070.28C
ATOM2006NEARGA1052−29.46965.7451.2101.0074.46N
ATOM2007CZARGA1052−30.74065.4491.5401.0075.81C
ATOM2008NH1ARGA1052−31.77266.1461.0761.0075.31N
ATOM2009NH2ARGA1052−30.99164.4302.3541.0078.34N
ATOM2010NASNA1053−30.85671.1080.0831.0067.63N
ATOM2011CAASNA1053−32.28971.3070.2991.0069.73C
ATOM2012CASNA1053−32.87669.9840.7191.0069.35C
ATOM2013OASNA1053−33.00569.075−0.0951.0069.80O
ATOM2014CBASNA1053−32.99671.829−0.9571.0070.79C
ATOM2015CGASNA1053−32.75973.310−1.2011.0072.54C
ATOM2016OD1ASNA1053−33.22373.847−2.1961.0078.00O
ATOM2017ND2ASNA1053−32.03773.975−0.2921.0081.34N
ATOM2018NTHRA1054−33.22869.8801.9961.0072.38N
ATOM2019CATHRA1054−33.75368.6472.5521.0072.53C
ATOM2020CTHRA1054−35.26668.5882.5101.0073.02C
ATOM2021OTHRA1054−35.82667.5162.3051.0077.21O
ATOM2022CBTHRA1054−33.25168.4583.9821.0071.32C
ATOM2023OG1THRA1054−33.55769.6194.7581.0071.59O
ATOM2024CG2THRA1054−31.74868.2543.9811.0073.10C
ATOM2025NASNA1055−35.90869.7372.7021.0075.24N
ATOM2026CAASNA1055−37.36069.8422.8151.0076.78C
ATOM2027CASNA1055−37.87669.0534.0461.0076.94C
ATOM2028OASNA1055−38.98568.5224.0441.0082.59O
ATOM2029CBASNA1055−38.06269.3981.5171.0076.21C
ATOM2030CGASNA1055−39.51169.8671.4311.0078.54C
ATOM2031OD1ASNA1055−39.93070.8122.1091.0077.71O
ATOM2032ND2ASNA1055−40.28569.1990.5871.0087.37N
ATOM2033NGLYA1056−37.05468.9845.0911.0076.49N
ATOM2034CAGLYA1056−37.40368.2836.3141.0076.60C
ATOM2035CGLYA1056−36.99566.8326.4091.0074.80C
ATOM2036OGLYA1056−37.02866.2987.4891.0077.03O
ATOM2037NVALA1057−36.61066.1925.3011.0074.21N
ATOM2038CAVALA1057−36.24664.7705.3201.0073.68C
ATOM2039CVALA1057−34.74564.5545.0951.0073.15C
ATOM2040OVALA1057−34.18165.1144.1511.0077.13O
ATOM2041CBVALA1057−37.02963.9814.2611.0072.56C
ATOM2042CG1VALA1057−36.71162.4984.3681.0074.94C
ATOM2043CG2VALA1057−38.52364.2014.4321.0070.86C
ATOM2044NILEA1058−34.12063.7425.9631.0071.83N
ATOM2045CAILEA1058−32.69863.3475.8431.0071.92C
ATOM2046CILEA1058−32.52061.8335.8721.0073.30C
ATOM2047OILEA1058−33.38261.1116.3701.0076.64O
ATOM2048CBILEA1058−31.81563.9536.9561.0067.96C
ATOM2049CG1ILEA1058−32.22063.4488.3641.0071.45C
ATOM2050CG2ILEA1058−31.88365.4576.8991.0065.81C
ATOM2051CD1ILEA1058−31.20563.8169.4451.0065.73C
ATOM2052NTHRA1059−31.39261.3625.3431.0072.25N
ATOM2053CATHRA1059−31.07059.9325.3451.0071.10C
ATOM2054CTHRA1059−30.53359.5456.7121.0070.11C
ATOM2055OTHRA1059−30.16660.4057.4951.0071.12O
ATOM2056CBTHRA1059−29.99059.5954.3531.0070.42C
ATOM2057OG1THRA1059−28.77560.2284.7761.0067.63O
ATOM2058CG2THRA1059−30.37760.0562.8741.0063.19C
ATOM2059NLYSA1060−30.49358.2446.9721.0070.88N
ATOM2060CALYSA1060−29.95057.6848.2081.0071.95C
ATOM2061CLYSA1060−28.48158.0878.4161.0072.94C
ATOM2062OLYSA1060−28.08258.4529.5361.0071.54O
ATOM2063CBLYSA1060−30.07456.1518.2151.0070.63C
ATOM2064CGLYSA1060−29.32555.4669.3871.0075.49C
ATOM2065CDLYSA1060−29.50053.9539.4191.0073.88C
ATOM2066CELYSA1060−28.42253.32210.3081.0071.79C
ATOM2067NZLYSA1060−28.58451.84810.4271.0080.16N
ATOM2068NASPA1061−27.70658.0107.3301.0068.84N
ATOM2069CAASPA1061−26.28258.3397.3201.0068.20C
ATOM2070CASPA1061−26.04359.8147.5861.0066.58C
ATOM2071OASPA1061−25.07660.1488.2551.0070.66O
ATOM2072CBASPA1061−25.62257.9235.9831.0064.64C
ATOM2073CGASPA1061−25.57256.3865.7881.0073.92C
ATOM2074OD1ASPA1061−25.50155.6196.7821.0083.01O
ATOM2075OD2ASPA1061−25.60355.9354.6301.0082.92O
ATOM2076NGLUA1062−26.90960.6877.0711.0060.10N
ATOM2077CAGLUA1062−26.85662.1137.4371.0062.56C
ATOM2078CGLUA1062−27.20362.3178.9551.0064.45C
ATOM2079OGLUA1062−26.55963.1279.6191.0062.28O
ATOM2080CBGLUA1062−27.77062.9106.5721.0060.74C
ATOM2081CGGLUA1062−27.24363.0185.1421.0060.93C
ATOM2082CDGLUA1062−28.23063.6064.1981.0068.90C
ATOM2083OE1GLUA1062−29.44663.4984.4881.0067.42O
ATOM2084OE2GLUA1062−27.79564.1823.1551.0073.08O
ATOM2085NALAA1063−28.19761.5909.4851.0064.40N
ATOM2086CAALAA1063−28.49461.64710.9551.0068.01C
ATOM2087CALAA1063−27.25161.29611.7481.0069.72C
ATOM2088OALAA1063−26.94861.94012.7451.0079.20O
ATOM2089CBALAA1063−29.63760.72311.3311.0065.57C
ATOM2090NGLUA1064−26.52960.26911.2981.0073.91N
ATOM2091CAGLUA1064−25.26159.87311.9201.0071.50C
ATOM2092CGLUA1064−24.21860.97311.8151.0072.04C
ATOM2093OGLUA1064−23.46561.15812.7471.0076.73O
ATOM2094CBGLUA1064−24.74658.58411.2941.0070.30C
ATOM2095CGGLUA1064−23.39158.09011.7721.0071.10C
ATOM2096CDGLUA1064−23.01256.71511.1951.0081.17C
ATOM2097OE1GLUA1064−23.71156.19410.2511.0087.16O
ATOM2098OE2GLUA1064−22.00656.15011.6881.0089.77O
ATOM2099NLYSA1065−24.16861.70110.6951.0073.67N
ATOM2100CALYSA1065−23.20462.82110.5681.0073.69C
ATOM2101CLYSA1065−23.46663.90311.6201.0070.10C
ATOM2102OLYSA1065−22.55364.32512.3101.0068.11O
ATOM2103CBLYSA1065−23.23263.4619.1901.0075.31C
ATOM2104CGLYSA1065−22.89162.5468.0221.0080.59C
ATOM2105CDLYSA1065−21.42262.2237.9381.0085.80C
ATOM2106CELYSA1065−21.09861.3796.6801.0081.37C
ATOM2107NZLYSA1065−21.66161.9445.4251.0072.55N
ATOM2108NLEUA1066−24.71864.33511.7231.0069.54N
ATOM2109CALEUA1066−25.13365.30512.7481.0071.70C
ATOM2110CLEUA1066−24.85464.80414.1651.0070.08C
ATOM2111OLEUA1066−24.47665.57215.0331.0068.08O
ATOM2112CBLEUA1066−26.62165.60512.6361.0071.78C
ATOM2113CGLEUA1066−27.05666.46611.4681.0076.18C
ATOM2114CD1LEUA1066−28.56566.35711.2611.0078.42C
ATOM2115CD2LEUA1066−26.62567.89911.7151.0077.95C
ATOM2116NPHEA1067−25.04963.51314.3731.0071.17N
ATOM2117CAPHEA1067−24.84462.89915.6731.0071.75C
ATOM2118CPHEA1067−23.38362.90216.1041.0071.56C
ATOM2119OPHEA1067−23.09863.17117.2751.0076.85O
ATOM2120CBPHEA1067−25.37861.48015.6691.0074.38C
ATOM2121CGPHEA1067−25.04660.74016.8801.0075.45C
ATOM2122CD1PHEA1067−25.81060.89718.0121.0078.29C
ATOM2123CD2PHEA1067−23.95759.87716.9091.0081.22C
ATOM2124CE1PHEA1067−25.50960.22119.1381.0077.84C
ATOM2125CE2PHEA1067−23.65059.19418.0431.0081.99C
ATOM2126CZPHEA1067−24.42559.36119.1651.0078.68C
ATOM2127NASNA1068−22.46462.61115.1881.0072.06N
ATOM2128CAASNA1068−21.03562.65815.5201.0072.87C
ATOM2129CASNA1068−20.61164.09215.8881.0074.75C
ATOM2130OASNA1068−19.72264.27216.7161.0076.41O
ATOM2131CBASNA1068−20.13462.15114.4001.0070.30C
ATOM2132CGASNA1068−20.47760.74113.9361.0077.74C
ATOM2133OD1ASNA1068−20.90259.90314.7221.0071.67O
ATOM2134ND2ASNA1068−20.28660.47912.6171.0072.67N
ATOM2135NGLNA1069−21.24465.09415.2751.0073.62N
ATOM2136CAGLNA1069−20.98266.48715.6311.0073.62C
ATOM2137CGLNA1069−21.54166.80317.0201.0073.06C
ATOM2138OGLNA1069−20.87467.45517.8021.0068.92O
ATOM2139CBGLNA1069−21.58667.44914.6341.0071.42C
ATOM2140CGGLNA1069−20.96767.37913.2491.0081.86C
ATOM2141CDGLNA1069−21.71768.23512.2391.0081.55C
ATOM2142OE1GLNA1069−22.50469.11612.6141.0087.86O
ATOM2143NE2GLNA1069−21.48067.98010.9481.0091.37N
ATOM2144NASPA1070−22.75966.34117.3041.0069.64N
ATOM2145CAASPA1070−23.36266.54918.6151.0070.15C
ATOM2146CASPA1070−22.51065.87919.6891.0070.56C
ATOM2147OASPA1070−22.27266.48620.7371.0071.26O
ATOM2148CBASPA1070−24.79566.03818.6641.0068.69C
ATOM2149CGASPA1070−25.74266.82817.7561.0077.13C
ATOM2150OD1ASPA1070−25.44467.99717.3921.0075.90O
ATOM2151OD2ASPA1070−26.79466.27717.4001.0074.40O
ATOM2152NVALA1071−22.05864.64919.4261.0067.27N
ATOM2153CAVALA1071−21.15063.94820.3411.0069.23C
ATOM2154CVALA1071−19.90664.78020.5771.0070.51C
ATOM2155OVALA1071−19.50964.96821.7141.0071.56O
ATOM2156CBVALA1071−20.73762.56319.8201.0066.16C
ATOM2157CG1VALA1071−19.56162.08620.5481.0063.68C
ATOM2158CG2VALA1071−21.88761.57019.9521.0068.51C
ATOM2159NASPA1072−19.29465.28019.5001.0074.44N
ATOM2160CAASPA1072−18.12866.15419.6231.0073.07C
ATOM2161CASPA1072−18.41067.32120.5551.0074.85C
ATOM2162OASPA1072−17.62867.58521.4641.0078.47O
ATOM2163CBASPA1072−17.68866.70318.2691.0077.99C
ATOM2164CGASPA1072−16.50267.64518.3871.0082.49C
ATOM2165OD1ASPA1072−15.35567.15618.5211.0093.79O
ATOM2166OD2ASPA1072−16.73068.87418.3411.0088.07O
ATOM2167NALAA1073−19.53068.00320.3161.0070.34N
ATOM2168CAALAA1073−19.98169.12621.1531.0071.13C
ATOM2169CALAA1073−20.17868.74222.6431.0072.31C
ATOM2170OALAA1073−19.77169.49523.5201.0072.64O
ATOM2171CBALAA1073−21.26169.67520.6201.0068.05C
ATOM2172NALAA1074−20.79767.58422.8901.0069.54N
ATOM2173CAALAA1074−21.08567.09624.2441.0071.93C
ATOM2174CALAA1074−19.79566.90325.0281.0072.36C
ATOM2175OALAA1074−19.68467.37826.1491.0077.33O
ATOM2176CBALAA1074−21.85165.80724.1931.0071.13C
ATOM2177NVALA1075−18.83666.20724.4151.0072.46N
ATOM2178CAVALA1075−17.51165.97324.9951.0074.25C
ATOM2179CVALA1075−16.75267.26225.3481.0074.70C
ATOM2180OVALA1075−16.16367.34526.4291.0074.40O
ATOM2181CBVALA1075−16.64865.10224.0731.0074.74C
ATOM2182CG1VALA1075−15.20865.07624.5391.0075.33C
ATOM2183CG2VALA1075−17.20563.69624.0331.0076.41C
ATOM2184NARGA1076−16.76868.24624.4551.0072.53N
ATOM2185CAARGA1076−16.12469.54624.7261.0074.21C
ATOM2186CARGA1076−16.76670.23025.9111.0074.53C
ATOM2187OARGA1076−16.07070.83826.7351.0074.38O
ATOM2188CBARGA1076−16.20470.48723.5331.0070.53C
ATOM2189CGARGA1076−15.35670.07022.3671.0076.54C
ATOM2190CDARGA1076−15.46571.07021.2051.0082.85C
ATOM2191NEARGA1076−15.06570.43019.9511.0091.84N
ATOM2192CZARGA1076−13.80270.18319.5691.0096.99C
ATOM2193NH1ARGA1076−12.74670.51720.3341.0097.76N
ATOM2194NH2ARGA1076−13.58069.58818.3931.00103.37N
ATOM2195NGLYA1077−18.09170.12025.9771.0072.90N
ATOM2196CAGLYA1077−18.86570.65627.0591.0073.02C
ATOM2197CGLYA1077−18.54369.99528.3921.0072.32C
ATOM2198OGLYA1077−18.32270.69029.3871.0073.12O
ATOM2199NILEA1078−18.52368.66128.3991.0072.41N
ATOM2200CAILEA1078−18.10467.85829.5641.0073.34C
ATOM2201CILEA1078−16.73968.29330.0831.0071.54C
ATOM2202OILEA1078−16.57768.52231.2801.0076.16O
ATOM2203CBILEA1078−18.04466.36329.2101.0074.38C
ATOM2204CG1ILEA1078−19.45365.75729.1981.0079.15C
ATOM2205CG2ILEA1078−17.20565.61630.1741.0064.66C
ATOM2206CD1ILEA1078−19.51464.38528.5541.0075.65C
ATOM2207NLEUA1079−15.77568.40729.1771.0072.22N
ATOM2208CALEUA1079−14.40768.81929.5121.0073.11C
ATOM2209CLEUA1079−14.27870.27530.0011.0073.92C
ATOM2210OLEUA1079−13.28170.61330.6271.0078.75O
ATOM2211CBLEUA1079−13.46268.57828.3171.0072.14C
ATOM2212CGLEUA1079−13.19267.11527.9091.0072.35C
ATOM2213CD1LEUA1079−12.30167.07426.6791.0059.40C
ATOM2214CD2LEUA1079−12.54366.30929.0311.0062.86C
ATOM2215NARGA1080−15.26971.11829.7141.0073.61N
ATOM2216CAARGA1080−15.33072.49030.2511.0074.19C
ATOM2217CARGA1080−16.03972.53731.6091.0072.82C
ATOM2218OARGA1080−15.89473.50532.3311.0072.73O
ATOM2219CBARGA1080−16.06173.43929.3011.0074.26C
ATOM2220CGARGA1080−15.23673.95528.1401.0079.49C
ATOM2221CDARGA1080−15.96475.12127.4601.0079.38C
ATOM2222NEARGA1080−17.26074.69126.9221.0088.39N
ATOM2223CZARGA1080−17.53574.38725.6361.0087.86C
ATOM2224NH1ARGA1080−16.60874.45124.6651.0087.22N
ATOM2225NH2ARGA1080−18.77774.00925.3141.0081.94N
ATOM2226NASNA1081−16.80071.49531.9461.0072.20N
ATOM2227CAASNA1081−17.55371.46433.1831.0070.14C
ATOM2228CASNA1081−16.68070.90334.2931.0070.99C
ATOM2229OASNA1081−16.29569.73334.2521.0070.87O
ATOM2230CBASNA1081−18.81770.64233.0021.0068.83C
ATOM2231CGASNA1081−19.78970.81334.1371.0074.52C
ATOM2232OD1ASNA1081−19.52170.40535.2751.0081.88O
ATOM2233ND2ASNA1081−20.93771.41733.8481.0065.69N
ATOM2234NALAA1082−16.37471.75035.2811.0069.81N
ATOM2235CAALAA1082−15.53971.38636.4591.0071.34C
ATOM2236CALAA1082−15.94270.10137.2031.0070.68C
ATOM2237OALAA1082−15.08169.42637.7481.0077.40O
ATOM2238CBALAA1082−15.50872.54637.4511.0064.36C
ATOM2239NLYSA1083−17.23369.78437.2131.0068.55N
ATOM2240CALYSA1083−17.77668.62337.9311.0069.05C
ATOM2241CLYSA1083−17.79867.33737.0931.0072.30C
ATOM2242OLYSA1083−17.73266.24337.6491.0076.42O
ATOM2243CBLYSA1083−19.19368.92338.3981.0070.45C
ATOM2244CGLYSA1083−19.35970.29739.0631.0075.92C
ATOM2245CDLYSA1083−20.72070.44739.6981.0081.44C
ATOM2246CELYSA1083−20.92671.86440.2171.0085.50C
ATOM2247NZLYSA1083−22.14871.99441.0681.0088.29N
ATOM2248NLEUA1084−17.89167.46035.7651.0068.40N
ATOM2249CALEUA1084−17.89866.28634.8941.0070.90C
ATOM2250CLEUA1084−16.51865.92434.4021.0070.17C
ATOM2251OLEUA1084−16.24764.74034.2051.0072.79O
ATOM2252CBLEUA1084−18.84366.49733.6941.0068.64C
ATOM2253CGLEUA1084−20.30466.80134.0511.0071.12C
ATOM2254CD1LEUA1084−21.21366.91632.7871.0059.19C
ATOM2255CD2LEUA1084−20.84765.74735.0101.0056.68C
ATOM2256NLYSA1085−15.65066.91734.2051.0068.34N
ATOM2257CALYSA1085−14.31666.66333.6521.0068.63C
ATOM2258CLYSA1085−13.53865.55934.3831.0068.76C
ATOM2259OLYSA1085−13.07064.64333.7231.0075.06O
ATOM2260CBLYSA1085−13.48267.95133.5651.0067.89C
ATOM2261CGLYSA1085−12.11067.77032.9171.0069.24C
ATOM2262CDLYSA1085−11.32269.05432.9251.0072.42C
ATOM2263CELYSA1085−10.04068.90532.1381.0077.84C
ATOM2264NZLYSA1085−9.13167.88132.7451.0085.14N
ATOM2265NPROA1086−13.39965.63635.7401.0069.24N
ATOM2266CAPROA1086−12.63764.58636.4401.0069.18C
ATOM2267CPROA1086−13.22663.19036.2611.0068.97C
ATOM2268OPROA1086−12.48862.19736.1991.0069.99O
ATOM2269CBPROA1086−12.77164.96637.9231.0067.72C
ATOM2270CGPROA1086−13.20166.35737.9501.0072.53C
ATOM2271CDPROA1086−13.91866.64636.6791.0066.17C
ATOM2272NVALA1087−14.54963.13636.1841.0068.84N
ATOM2273CAVALA1087−15.26561.88536.0921.0072.28C
ATOM2274CVALA1087−14.98561.29134.7431.0073.91C
ATOM2275OVALA1087−14.48060.17634.6721.0077.11O
ATOM2276CBVALA1087−16.76962.06236.3051.0072.36C
ATOM2277CG1VALA1087−17.43160.72036.3681.0078.50C
ATOM2278CG2VALA1087−17.03262.83237.6021.0075.48C
ATOM2279NTYRA1088−15.31062.04733.6911.0076.42N
ATOM2280CATYRA1088−15.08761.66032.2901.0074.22C
ATOM2281CTYRA1088−13.65561.24132.0541.0073.79C
ATOM2282OTYRA1088−13.41460.18431.5021.0071.26O
ATOM2283CBTYRA1088−15.42062.84031.3821.0076.80C
ATOM2284CGTYRA1088−15.34562.53629.9091.0078.30C
ATOM2285CD1TYRA1088−16.44761.99429.2251.0082.88C
ATOM2286CD2TYRA1088−14.18162.78729.1851.0082.17C
ATOM2287CE1TYRA1088−16.36861.71227.8381.0078.90C
ATOM2288CE2TYRA1088−14.10262.51127.8231.0073.71C
ATOM2289CZTYRA1088−15.19961.97627.1621.0071.96C
ATOM2290OHTYRA1088−15.09961.70525.8041.0090.19O
ATOM2291NASPA1089−12.70862.08232.4831.0073.28N
ATOM2292CAASPA1089−11.26261.78032.3991.0072.57C
ATOM2293CASPA1089−10.88060.40332.9361.0071.20C
ATOM2294OASPA1089−9.98759.75532.4071.0070.55O
ATOM2295CBASPA1089−10.43862.80233.2081.0072.85C
ATOM2296CGASPA1089−10.16264.11832.4681.0079.93C
ATOM2297OD1ASPA1089−10.55164.28731.2741.0078.98O
ATOM2298OD2ASPA1089−9.52865.00433.1201.0069.59O
ATOM2299NSERA1090−11.56259.96933.9931.0072.25N
ATOM2300CASERA1090−11.24458.71134.6701.0071.13C
ATOM2301CSERA1090−11.89357.49534.0271.0069.01C
ATOM2302OSERA1090−11.41156.38334.1991.0069.79O
ATOM2303CBSERA1090−11.68458.79136.1241.0067.58C
ATOM2304OGSERA1090−13.08558.66536.2321.0065.84O
ATOM2305NLEUA1091−12.97957.70333.2931.0072.26N
ATOM2306CALEUA1091−13.72756.59332.7291.0070.30C
ATOM2307CLEUA1091−13.09256.02331.4621.0073.59C
ATOM2308OLEUA1091−12.42056.71530.7051.0075.14O
ATOM2309CBLEUA1091−15.17257.01432.4571.0073.21C
ATOM2310CGLEUA1091−16.06057.29833.6871.0069.70C
ATOM2311CD1LEUA1091−17.37457.86933.2111.0074.59C
ATOM2312CD2LEUA1091−16.28756.05134.4821.0061.23C
ATOM2313NASPA1092−13.32454.73531.2571.0073.36N
ATOM2314CAASPA1092−12.95554.04630.0171.0074.54C
ATOM2315CASPA1092−13.91054.48028.9271.0072.07C
ATOM2316OASPA1092−14.93855.08829.2301.0069.09O
ATOM2317CBASPA1092−13.09352.54730.1801.0074.48C
ATOM2318CGASPA1092−14.50252.15730.5221.0082.40C
ATOM2319OD1ASPA1092−15.39352.05929.6431.0078.10O
ATOM2320OD2ASPA1092−14.70251.95631.7021.0093.39O
ATOM2321NALAA1093−13.56154.15627.6771.0071.26N
ATOM2322CAALAA1093−14.33254.53026.4551.0072.91C
ATOM2323CALAA1093−15.81754.18526.4821.0072.49C
ATOM2324OALAA1093−16.64255.01626.1431.0077.77O
ATOM2325CBALAA1093−13.68253.88425.2131.0068.92C
ATOM2326NVALA1094−16.15252.95926.8821.0074.25N
ATOM2327CAVALA1094−17.56652.51726.9701.0076.09C
ATOM2328CVALA1094−18.38853.29728.0211.0074.99C
ATOM2329OVALA1094−19.55053.68627.7601.0074.98O
ATOM2330CBVALA1094−17.68051.01427.2681.0077.56C
ATOM2331CG1VALA1094−19.12450.63427.5181.0080.02C
ATOM2332CG2VALA1094−17.09150.19226.0981.0077.82C
ATOM2333NARGA1095−17.80453.52729.1921.0074.55N
ATOM2334CAARGA1095−18.50254.30630.2451.0072.87C
ATOM2335CARGA1095−18.56455.79229.8771.0070.86C
ATOM2336OARGA1095−19.52956.47130.2241.0070.60O
ATOM2337CBARGA1095−17.85554.09031.5981.0074.90C
ATOM2338CGARGA1095−17.94452.64832.0491.0074.88C
ATOM2339CDARGA1095−17.45652.51933.4651.0076.13C
ATOM2340NEARGA1095−17.76551.22234.0391.0068.67N
ATOM2341CZARGA1095−17.03550.11133.9351.0075.88C
ATOM2342NH1ARGA1095−15.90050.07733.2611.0077.10N
ATOM2343NH2ARGA1095−17.45648.99934.5231.0078.82N
ATOM2344NARGA1096−17.55056.30429.1761.0070.40N
ATOM2345CAARGA1096−17.64657.65428.6161.0072.78C
ATOM2346CARGA1096−18.89557.78127.6831.0075.95C
ATOM2347OARGA1096−19.60358.79027.7411.0076.20O
ATOM2348CBARGA1096−16.38258.03227.8491.0074.96C
ATOM2349CGARGA1096−15.21258.38128.7291.0076.80C
ATOM2350CDARGA1096−13.98758.69927.8881.0073.69C
ATOM2351NEARGA1096−12.82158.87728.7241.0075.05N
ATOM2352CZARGA1096−11.61759.27628.3341.0076.09C
ATOM2353NH1ARGA1096−11.35859.56827.0621.0079.66N
ATOM2354NH2ARGA1096−10.64259.38829.2441.0073.76N
ATOM2355NALAA1097−19.15456.76726.8421.0074.52N
ATOM2356CAALAA1097−20.38156.76225.9931.0075.40C
ATOM2357CALAA1097−21.60656.78726.8991.0074.68C
ATOM2358OALAA1097−22.59657.44426.5941.0075.19O
ATOM2359CBALAA1097−20.42855.55625.0841.0065.57C
ATOM2360NALAA1098−21.53856.06828.0191.0077.29N
ATOM2361CAALAA1098−22.62756.11029.0041.0077.63C
ATOM2362CALAA1098−22.85557.54929.4251.0075.46C
ATOM2363OALAA1098−23.95358.03729.3061.0077.90O
ATOM2364CBALAA1098−22.31955.25230.2011.0072.80C
ATOM2365NLEUA1099−21.80858.21529.9061.0074.27N
ATOM2366CALEUA1099−21.93559.61030.3891.0073.67C
ATOM2367CLEUA1099−22.49360.54229.3291.0073.73C
ATOM2368OLEUA1099−23.41261.32829.6021.0073.92O
ATOM2369CBLEUA1099−20.59060.14430.8861.0076.16C
ATOM2370CGLEUA1099−20.61261.38131.7941.0076.75C
ATOM2371CD1LEUA1099−21.49561.16733.0001.0072.22C
ATOM2372CD2LEUA1099−19.18761.74132.2361.0066.69C
ATOM2373NILEA1100−21.94160.45428.1151.0072.03N
ATOM2374CAILEA1100−22.43061.25226.9861.0070.39C
ATOM2375CILEA1100−23.90860.99826.7581.0069.34C
ATOM2376OILEA1100−24.67461.94126.5521.0070.67O
ATOM2377CBILEA1100−21.67260.96425.6771.0074.51C
ATOM2378CG1ILEA1100−20.20861.41125.7891.0074.12C
ATOM2379CG2ILEA1100−22.36861.68424.5231.0066.55C
ATOM2380CD1ILEA1100−19.27360.64324.9101.0071.96C
ATOM2381NASNA1101−24.30559.72726.7981.0069.64N
ATOM2382CAASNA1101−25.71659.34026.6561.0071.15C
ATOM2383CASNA1101−26.58760.15427.5961.0073.26C
ATOM2384OASNA1101−27.60660.72027.1581.0073.01O
ATOM2385CBASNA1101−25.85957.81926.8801.0074.91C
ATOM2386CGASNA1101−27.24257.28626.6211.0072.41C
ATOM2387OD1ASNA1101−28.21857.89526.9741.0068.21O
ATOM2388ND2ASNA1101−27.32156.11025.9951.0066.91N
ATOM2389NMETA1102−26.19160.21728.8761.0073.18N
ATOM2390CAMETA1102−26.92260.98629.8941.0073.53C
ATOM2391CMETA1102−26.89762.47229.5791.0072.72C
ATOM2392OMETA1102−27.90963.13029.7191.0075.34O
ATOM2393CBMETA1102−26.34160.78831.3151.0069.92C
ATOM2394CGMETA1102−26.31159.34331.8461.0071.61C
ATOM2395SDMETA1102−25.63959.20533.5581.0075.01S
ATOM2396CEMETA1102−26.90360.13334.4361.0072.22C
ATOM2397NVALA1103−25.74362.99729.1601.0073.08N
ATOM2398CAVALA1103−25.65064.43028.7741.0075.10C
ATOM2399CVALA1103−26.60564.73627.5851.0074.50C
ATOM2400OVALA1103−27.28065.76927.5871.0074.33O
ATOM2401CBVALA1103−24.20364.85928.4451.0074.63C
ATOM2402CG1VALA1103−24.18166.21427.7731.0074.66C
ATOM2403CG2VALA1103−23.34564.89229.7151.0073.28C
ATOM2404NPHEA1104−26.64863.83826.5981.0072.50N
ATOM2405CAPHEA1104−27.64163.93725.5071.0077.14C
ATOM2406CPHEA1104−29.05064.13326.0871.0077.59C
ATOM2407OPHEA1104−29.74665.02325.6841.0078.04O
ATOM2408CBPHEA1104−27.61562.71224.5571.0078.31C
ATOM2409CGPHEA1104−26.87062.93323.2591.0081.99C
ATOM2410CD1PHEA1104−25.48963.02923.2381.0078.79C
ATOM2411CD2PHEA1104−27.58063.03722.0271.0090.20C
ATOM2412CE1PHEA1104−24.80063.22722.0211.0077.16C
ATOM2413CE2PHEA1104−26.91063.23720.8211.0084.00C
ATOM2414CZPHEA1104−25.50963.33220.8231.0086.09C
ATOM2415NGLNA1105−29.44663.29627.0391.0076.01N
ATOM2416CAGLNA1105−30.81863.33527.5751.0075.44C
ATOM2417CGLNA1105−31.15964.48028.5441.0074.63C
ATOM2418OGLNA1105−32.24965.04328.4551.0075.26O
ATOM2419CBGLNA1105−31.10062.01228.2701.0077.85C
ATOM2420CGGLNA1105−32.56761.75728.6151.0075.54C
ATOM2421CDGLNA1105−32.78160.37829.1681.0075.71C
ATOM2422OE1GLNA1105−31.83459.62029.3401.0071.29O
ATOM2423NE2GLNA1105−34.02560.03929.4501.0070.96N
ATOM2424NMETA1106−30.25064.81929.4561.0076.62N
ATOM2425CAMETA1106−30.52965.83830.5241.0077.20C
ATOM2426CMETA1106−29.66667.10030.4981.0075.43C
ATOM2427OMETA1106−29.90168.01031.2961.0078.05O
ATOM2428CBMETA1106−30.31965.21631.9021.0079.39C
ATOM2429CGMETA1106−30.88663.83232.1241.0085.85C
ATOM2430SDMETA1106−30.09163.06433.5421.0085.72S
ATOM2431CEMETA1106−29.93361.41932.9171.0081.10C
ATOM2432NGLYA1107−28.67567.16629.6091.0075.52N
ATOM2433CAGLYA1107−27.74868.29429.5601.0076.26C
ATOM2434CGLYA1107−26.70268.16630.6511.0078.60C
ATOM2435OGLYA1107−26.82767.33031.5551.0078.11O
ATOM2436NGLUA1108−25.66668.99330.5731.0079.86N
ATOM2437CAGLUA1108−24.55668.93131.5481.0082.66C
ATOM2438CGLUA1108−24.96669.22432.9661.0082.97C
ATOM2439OGLUA1108−24.49568.56033.8911.0086.76O
ATOM2440CBGLUA1108−23.45469.90131.1851.0082.78C
ATOM2441CGGLUA1108−22.71469.51229.9691.0086.49C
ATOM2442CDGLUA1108−21.72270.55629.5541.0090.68C
ATOM2443OE1GLUA1108−21.14871.27130.4091.0090.93O
ATOM2444OE2GLUA1108−21.52370.65928.3481.00103.73O
ATOM2445NTHRA1109−25.83770.21433.1321.0082.33N
ATOM2446CATHRA1109−26.32470.59734.4511.0080.83C
ATOM2447CTHRA1109−27.06169.45535.1121.0079.03C
ATOM2448OTHRA1109−26.75569.09836.2471.0084.70O
ATOM2449CBTHRA1109−27.27071.80234.3861.0079.62C
ATOM2450OG1THRA1109−26.59172.90733.7761.0080.39O
ATOM2451CG2THRA1109−27.73172.19635.7901.0081.01C
ATOM2452NGLYA1110−28.02868.88934.3951.0078.14N
ATOM2453CAGLYA1110−28.79467.74934.8881.0077.59C
ATOM2454CGLYA1110−27.89366.63735.3991.0076.91C
ATOM2455OGLYA1110−28.14766.06936.4501.0077.84O
ATOM2456NVALA1111−26.83866.34834.6391.0074.81N
ATOM2457CAVALA1111−25.90165.27434.9521.0073.76C
ATOM2458CVALA1111−24.90165.66336.0631.0074.74C
ATOM2459OVALA1111−24.47564.80936.8631.0070.56O
ATOM2460CBVALA1111−25.09464.84433.7151.0068.49C
ATOM2461CG1VALA1111−24.17363.69534.0811.0064.99C
ATOM2462CG2VALA1111−26.02364.43832.5821.0068.09C
ATOM2463NALAA1112−24.53166.94936.0961.0074.29N
ATOM2464CAALAA1112−23.63167.49137.1191.0077.63C
ATOM2465CALAA1112−24.25567.42338.4961.0077.53C
ATOM2466OALAA1112−23.54267.41039.4831.0082.24O
ATOM2467CBALAA1112−23.25668.93136.7981.0077.58C
ATOM2468NGLYA1113−25.59167.38138.5531.0079.76N
ATOM2469CAGLYA1113−26.32367.27039.8081.0077.64C
ATOM2470CGLYA1113−26.26765.90040.4611.0078.25C
ATOM2471OGLYA1113−26.68765.78041.5881.0076.50O
ATOM2472NPHEA1114−25.75764.86139.7741.0077.88N
ATOM2473CAPHEA1114−25.58663.53340.3831.0076.03C
ATOM2474CPHEA1114−24.27663.50041.1791.0075.36C
ATOM2475OPHEA1114−23.39962.69340.8871.0073.89O
ATOM2476CBPHEA1114−25.55962.43739.3101.0081.97C
ATOM2477CGPHEA1114−26.84562.24738.5941.0084.39C
ATOM2478CD1PHEA1114−27.90661.60939.2021.0093.33C
ATOM2479CD2PHEA1114−27.00162.69637.3101.0090.99C
ATOM2480CE1PHEA1114−29.10861.43338.5211.0091.37C
ATOM2481CE2PHEA1114−28.18762.52636.6271.0091.29C
ATOM2482CZPHEA1114−29.24461.89437.2291.0089.69C
ATOM2483NTHRA1115−24.13964.36442.1861.0070.77N
ATOM2484CATHRA1115−22.86064.55042.8451.0071.53C
ATOM2485CTHRA1115−22.29263.25643.4201.0072.52C
ATOM2486OTHRA1115−21.12462.96443.2061.0074.92O
ATOM2487CBTHRA1115−22.95065.61343.9571.0074.18C
ATOM2488OG1THRA1115−23.63866.76143.4541.0081.29O
ATOM2489CG2THRA1115−21.56266.02744.4191.0065.46C
ATOM2490NASNA1116−23.11262.49444.1381.0073.53N
ATOM2491CAASNA1116−22.64461.26044.7791.0074.43C
ATOM2492CASNA1116−22.32760.17643.7531.0074.36C
ATOM2493OASNA1116−21.28659.52343.8541.0073.98O
ATOM2494CBASNA1116−23.65260.72345.8031.0076.08C
ATOM2495CGASNA1116−23.91661.70146.9581.0081.08C
ATOM2496OD1ASNA1116−22.99462.12547.6471.0076.24O
ATOM2497ND2ASNA1116−25.18162.05047.1631.0082.05N
ATOM2498NSERA1117−23.21859.98642.7761.0071.66N
ATOM2499CASERA1117−22.99059.01441.6971.0071.35C
ATOM2500CSERA1117−21.73659.30740.8951.0072.60C
ATOM2501OSERA1117−21.01058.38640.5671.0073.79O
ATOM2502CBSERA1117−24.17058.96240.7481.0074.52C
ATOM2503OGSERA1117−25.27158.32941.3511.0074.10O
ATOM2504NLEUA1118−21.48460.58040.5861.0074.22N
ATOM2505CALEUA1118−20.25360.99739.8631.0074.12C
ATOM2506CLEUA1118−18.96160.66040.6181.0073.96C
ATOM2507OLEUA1118−17.98460.19540.0251.0078.17O
ATOM2508CBLEUA1118−20.29962.50339.5661.0074.92C
ATOM2509CGLEUA1118−21.33762.93738.5001.0077.06C
ATOM2510CD1LEUA1118−21.51664.48438.5181.0079.91C
ATOM2511CD2LEUA1118−20.95062.45637.1201.0079.15C
ATOM2512NARGA1119−18.97260.90141.9171.0073.81N
ATOM2513CAARGA1119−17.85760.57142.8061.0076.37C
ATOM2514CARGA1119−17.58659.06942.8371.0073.72C
ATOM2515OARGA1119−16.43958.63342.7601.0071.54O
ATOM2516CBARGA1119−18.17961.04144.2291.0072.72C
ATOM2517CGARGA1119−17.06760.87345.2411.0081.26C
ATOM2518CDARGA1119−17.60860.97846.7121.0088.03C
ATOM2519NEARGA1119−18.62262.02746.8761.0097.80N
ATOM2520CZARGA1119−18.38763.35046.8521.00107.35C
ATOM2521NH1ARGA1119−17.14963.84946.6661.00110.91N
ATOM2522NH2ARGA1119−19.40764.20747.0161.00108.55N
ATOM2523NMETA1120−18.65358.29642.9581.0072.40N
ATOM2524CAMETA1120−18.55456.84342.9481.0075.44C
ATOM2525CMETA1120−18.05956.32341.6101.0072.59C
ATOM2526OMETA1120−17.19955.45541.5801.0074.45O
ATOM2527CBMETA1120−19.88456.22843.3381.0074.73C
ATOM2528CGMETA1120−20.11656.40744.8611.0079.31C
ATOM2529SDMETA1120−21.69855.81045.3411.0086.73S
ATOM2530CEMETA1120−22.84356.94344.5711.0082.05C
ATOM2531NLEUA1121−18.59256.85140.5161.0070.51N
ATOM2532CALEUA1121−18.06756.49539.2111.0073.43C
ATOM2533CLEUA1121−16.58156.81639.1491.0071.65C
ATOM2534OLEUA1121−15.81055.97038.7281.0074.87O
ATOM2535CBLEUA1121−18.80257.21338.0861.0067.49C
ATOM2536CGLEUA1121−20.24756.73437.8771.0072.80C
ATOM2537CD1LEUA1121−20.99057.72436.9681.0065.17C
ATOM2538CD2LEUA1121−20.34355.24137.3641.0062.04C
ATOM2539NGLNA1122−16.18658.02039.5651.0069.51N
ATOM2540CAGLNA1122−14.76058.41939.5511.0069.43C
ATOM2541CGLNA1122−13.85257.42240.3021.0069.96C
ATOM2542OGLNA1122−12.72657.18839.8891.0072.67O
ATOM2543CBGLNA1122−14.60259.82840.1231.0069.75C
ATOM2544CGGLNA1122−13.20360.43239.9751.0073.87C
ATOM2545CDGLNA1122−13.12561.88340.4431.0079.31C
ATOM2546OE1GLNA1122−14.13962.50540.7581.0088.60O
ATOM2547NE2GLNA1122−11.90762.42640.4901.0084.85N
ATOM2548NGLNA1123−14.36456.85041.3951.0066.33N
ATOM2549CAGLNA1123−13.65955.83342.1821.0071.40C
ATOM2550CGLNA1123−13.85754.37341.6881.0070.37C
ATOM2551OGLNA1123−13.32353.45042.3001.0068.04O
ATOM2552CBGLNA1123−14.08355.95843.6621.0066.15C
ATOM2553CGGLNA1123−13.70157.31844.2671.0065.21C
ATOM2554CDGLNA1123−14.26757.56645.6521.0069.71C
ATOM2555OE1GLNA1123−15.29457.01846.0351.0077.32O
ATOM2556NE2GLNA1123−13.58558.40446.4111.0063.29N
ATOM2557NLYSA1124−14.60954.17440.6041.0069.50N
ATOM2558CALYSA1124−14.89652.83040.0661.0074.09C
ATOM2559CLYSA1124−15.71951.95941.0481.0075.70C
ATOM2560OLYSA1124−15.56250.73341.0841.0075.06O
ATOM2561CBLYSA1124−13.60352.08939.6881.0079.02C
ATOM2562CGLYSA1124−12.57152.89238.8921.0083.65C
ATOM2563CDLYSA1124−12.68252.70137.3991.0089.63C
ATOM2564CELYSA1124−11.97853.75836.5401.0092.96C
ATOM2565NZLYSA1124−12.83355.01136.4161.00104.91N
ATOM2566NARGA1125−16.58652.60941.8281.0074.41N
ATOM2567CAARGA1125−17.48951.95842.7581.0073.39C
ATOM2568CARGA1125−18.78751.86241.9751.0072.92C
ATOM2569OARGA1125−19.75552.56942.2381.0075.78O
ATOM2570CBARGA1125−17.62552.76544.0491.0072.14C
ATOM2571CGARGA1125−16.30452.95944.7761.0074.10C
ATOM2572CDARGA1125−16.44853.86045.9741.0076.04C
ATOM2573NEARGA1125−17.14453.22647.0961.0083.94N
ATOM2574CZARGA1125−17.56753.85948.2031.0087.62C
ATOM2575NH1ARGA1125−17.37355.17948.3651.0091.97N
ATOM2576NH2ARGA1125−18.19553.17149.1721.0084.34N
ATOM2577NTRPA1126−18.77850.96341.0051.0074.20N
ATOM2578CATRPA1126−19.83950.84739.9811.0072.97C
ATOM2579CTRPA1126−21.22050.50740.5411.0071.90C
ATOM2580OTRPA1126−22.20351.18740.2321.0067.72O
ATOM2581CBTRPA1126−19.47349.78138.9591.0069.76C
ATOM2582CGTRPA1126−18.12449.89438.3351.0070.87C
ATOM2583CD1TRPA1126−17.18348.91738.2551.0066.79C
ATOM2584CD2TRPA1126−17.56451.04737.6991.0070.96C
ATOM2585NE1TRPA1126−16.06849.38637.6101.0069.94N
ATOM2586CE2TRPA1126−16.28050.69237.2601.0070.31C
ATOM2587CE3TRPA1126−18.02652.35137.4571.0071.68C
ATOM2588CZ2TRPA1126−15.46051.57636.6001.0074.24C
ATOM2589CZ3TRPA1126−17.20553.23536.8011.0071.20C
ATOM2590CH2TRPA1126−15.93952.85036.3791.0073.75C
ATOM2591NASPA1127−21.27349.45841.3571.0072.11N
ATOM2592CAASPA1127−22.53749.01241.9941.0074.28C
ATOM2593CASPA1127−23.16850.10042.8301.0072.96C
ATOM2594OASPA1127−24.37750.28342.7811.0077.35O
ATOM2595CBASPA1127−22.32647.76842.8741.0073.85C
ATOM2596CGASPA1127−22.17746.47542.0651.0080.36C
ATOM2597OD1ASPA1127−22.03446.53040.8361.0081.11O
ATOM2598OD2ASPA1127−22.20445.38642.6811.0097.24O
ATOM2599NGLUA1128−22.34950.81343.5861.0070.74N
ATOM2600CAGLUA1128−22.84051.88644.4521.0072.33C
ATOM2601CGLUA1128−23.29953.08943.6461.0070.63C
ATOM2602OGLUA1128−24.27653.74044.0171.0070.95O
ATOM2603CBGLUA1128−21.76452.29345.4201.0070.41C
ATOM2604CGGLUA1128−21.38451.18646.3641.0079.81C
ATOM2605CDGLUA1128−20.23251.57047.2191.0081.59C
ATOM2606OE1GLUA1128−20.44552.47648.0591.0090.24O
ATOM2607OE2GLUA1128−19.13150.96947.0531.0085.83O
ATOM2608NALAA1129−22.58753.37242.5611.0067.55N
ATOM2609CAALAA1129−23.00054.37541.5741.0071.98C
ATOM2610CALAA1129−24.37854.00041.0581.0070.13C
ATOM2611OALAA1129−25.26354.82741.0571.0076.48O
ATOM2612CBALAA1129−21.98954.46240.4101.0065.05C
ATOM2613NALAA1130−24.53452.74240.6341.0072.74N
ATOM2614CAALAA1130−25.82152.18240.1261.0070.99C
ATOM2615CALAA1130−26.95652.24941.1421.0070.01C
ATOM2616OALAA1130−28.12252.48740.7761.0068.38O
ATOM2617CBALAA1130−25.62350.73539.6761.0066.12C
ATOM2618NVALA1131−26.63252.03942.4161.0068.58N
ATOM2619CAVALA1131−27.66352.12443.4731.0067.85C
ATOM2620CVALA1131−28.22253.53543.5031.0069.11C
ATOM2621OVALA1131−29.44553.71943.5511.0067.62O
ATOM2622CBVALA1131−27.14951.68544.8511.0069.41C
ATOM2623CG1VALA1131−28.07052.23646.0241.0060.58C
ATOM2624CG2VALA1131−27.01650.14744.8861.0064.52C
ATOM2625NASNA1132−27.32354.51743.4661.0071.57N
ATOM2626CAASNA1132−27.70655.93543.5101.0073.21C
ATOM2627CASNA1132−28.40856.41242.2321.0070.74C
ATOM2628OASNA1132−29.42257.10442.3071.0068.21O
ATOM2629CBASNA1132−26.48656.81743.8281.0072.85C
ATOM2630CGASNA1132−26.11256.77645.2961.0078.49C
ATOM2631OD1ASNA1132−26.08857.80745.9621.0083.67O
ATOM2632ND2ASNA1132−25.82255.58845.8081.0084.32N
ATOM2633NLEUA1133−27.86956.04041.0761.0069.80N
ATOM2634CALEUA1133−28.46556.42139.7981.0072.25C
ATOM2635CLEUA1133−29.88355.85939.6331.0073.77C
ATOM2636OLEUA1133−30.74856.52839.0651.0078.51O
ATOM2637CBLEUA1133−27.56955.97138.6371.0074.37C
ATOM2638CGLEUA1133−26.18656.63238.5651.0074.55C
ATOM2639CD1LEUA1133−25.32756.04537.4361.0081.95C
ATOM2640CD2LEUA1133−26.33658.13938.4081.0079.93C
ATOM2641NALAA1134−30.12154.63940.1321.0072.16N
ATOM2642CAALAA1134−31.45554.02240.0901.0071.47C
ATOM2643CALAA1134−32.50554.77640.9501.0072.41C
ATOM2644OALAA1134−33.70154.58640.7411.0070.87O
ATOM2645CBALAA1134−31.37852.53340.5111.0068.95C
ATOM2646NLYSA1135−32.05255.62041.9011.0068.74N
ATOM2647CALYSA1135−32.94656.43642.7731.0072.99C
ATOM2648CLYSA1135−33.26657.85742.2311.0072.10C
ATOM2649OLYSA1135−33.58558.76243.0001.0074.92O
ATOM2650CBLYSA1135−32.34356.53644.1991.0069.32C
ATOM2651CGLYSA1135−32.40255.25345.0121.0076.21C
ATOM2652CDLYSA1135−31.67455.41046.3671.0077.13C
ATOM2653CELYSA1135−31.95054.24547.3101.0077.91C
ATOM2654NZLYSA1135−30.93854.13448.4341.0077.31N
ATOM2655NSERA1136−33.18558.05140.9161.0076.65N
ATOM2656CASERA1136−33.40459.36440.2981.0074.00C
ATOM2657CSERA1136−34.75359.51639.5941.0076.99C
ATOM2658OSERA1136−35.46458.53639.3201.0079.35O
ATOM2659CBSERA1136−32.28959.63539.3041.0075.82C
ATOM2660OGSERA1136−32.13958.57338.3681.0074.98O
ATOM2661NARGA1137−35.08160.77539.3131.0074.80N
ATOM2662CAARGA1137−36.21661.14138.4851.0072.91C
ATOM2663CARGA1137−35.96560.63937.0431.0071.62C
ATOM2664OARGA1137−36.88460.31236.3251.0070.32O
ATOM2665CBARGA1137−36.39562.66138.5041.0071.58C
ATOM2666CGARGA1137−37.56763.22037.7021.0076.14C
ATOM2667CDARGA1137−38.89662.71538.2311.0084.99C
ATOM2668NEARGA1137−40.03463.26737.4971.0085.80N
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ATOM2670NH1ARGA1137−41.64061.95138.5471.0074.66N
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ATOM2672NTRPA1138−34.70360.59236.6511.0070.67N
ATOM2673CATRPA1138−34.28960.02335.3921.0072.64C
ATOM2674CTRPA1138−34.75958.56935.2481.0074.44C
ATOM2675OTRPA1138−35.42858.23334.2791.0074.91O
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ATOM2677CGTRPA1138−32.16659.43434.1641.0072.19C
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ATOM2679CD2TRPA1138−31.02458.57434.2071.0068.92C
ATOM2680NE1TRPA1138−31.72458.71332.0921.0073.58N
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ATOM2682CE3TRPA1138−30.19058.12535.2231.0066.16C
ATOM2683CZ2TRPA1138−29.74957.29332.5701.0069.41C
ATOM2684CZ3TRPA1138−29.14257.26234.8931.0071.01C
ATOM2685CH2TRPA1138−28.93456.86333.6001.0071.24C
ATOM2686NTYRA1139−34.40757.72736.2121.0073.44N
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ATOM2688CTYRA1139−36.33756.17036.3971.0071.38C
ATOM2689OTYRA1139−36.94055.21835.9221.0072.03O
ATOM2690CBTYRA1139−34.06455.51637.2581.0074.06C
ATOM2691CGTYRA1139−34.53054.09537.4041.0070.82C
ATOM2692CD1TYRA1139−34.01253.10136.6011.0076.39C
ATOM2693CD2TYRA1139−35.49653.74638.3511.0076.13C
ATOM2694CE1TYRA1139−34.42951.79336.7201.0073.02C
ATOM2695CE2TYRA1139−35.92352.43638.4811.0079.81C
ATOM2696CZTYRA1139−35.37651.46137.6511.0077.76C
ATOM2697OHTYRA1139−35.77250.15437.7471.0078.31O
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ATOM2700CASNA1140−39.17557.39136.0281.0070.46C
ATOM2701OASNA1140−40.30756.92935.8821.0072.15O
ATOM2702CBASNA1140−38.75558.17238.3381.0068.60C
ATOM2703CGASNA1140−40.23058.24038.5721.0072.49C
ATOM2704OD1ASNA1140−40.86459.26438.3351.0072.97O
ATOM2705ND2ASNA1140−40.80157.13839.0401.0083.45N
ATOM2706NGLNA1141−38.57358.14535.1111.0071.15N
ATOM2707CAGLNA1141−39.24858.61633.8961.0071.46C
ATOM2708CGLNA1141−38.91957.75532.6771.0072.46C
ATOM2709OGLNA1141−39.82857.37831.9301.0069.45O
ATOM2710CBGLNA1141−38.88760.09133.6631.0071.54C
ATOM2711CGGLNA1141−39.49361.04034.6941.0074.06C
ATOM2712CDGLNA1141−40.98761.14234.5761.0084.66C
ATOM2713OE1GLNA1141−41.72660.86735.5391.0072.30O
ATOM2714NE2GLNA1141−41.46161.53533.3871.0078.96N
ATOM2715NTHRA1142−37.63357.45432.4901.0069.68N
ATOM2716CATHRA1142−37.16756.58231.4091.0071.24C
ATOM2717CTHRA1142−36.39955.38231.9971.0071.57C
ATOM2718OTHRA1142−35.17755.27431.7831.0073.67O
ATOM2719CBTHRA1142−36.31857.37330.3961.0073.10C
ATOM2720OG1THRA1142−35.33558.13431.1001.0065.73O
ATOM2721CG2THRA1142−37.22358.32629.5691.0063.92C
ATOM2722NPROA1143−37.12054.47532.7441.0068.91N
ATOM2723CAPROA1143−36.53753.29933.4041.0067.95C
ATOM2724CPROA1143−35.76252.37232.4971.0071.67C
ATOM2725OPROA1143−34.63852.01832.8341.0070.47O
ATOM2726CBPROA1143−37.76052.55333.9371.0070.48C
ATOM2727CGPROA1143−38.92053.13733.2521.0070.91C
ATOM2728CDPROA1143−38.57054.52833.0011.0068.98C
ATOM2729NASNA1144−36.35751.99331.3611.0070.87N
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ATOM2731CASNA1144−34.38251.61329.9461.0072.08C
ATOM2732OASNA1144−33.36950.92130.0711.0071.77O
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ATOM2734CGASNA1144−37.85950.07729.5151.0074.27C
ATOM2735OD1ASNA1144−37.91949.32330.4801.0076.06O
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ATOM2737NARGA1145−34.38852.83729.4091.0070.29N
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ATOM2739CARGA1145−32.14253.62730.1321.0070.79C
ATOM2740OARGA1145−30.95053.29929.9841.0071.02O
ATOM2741CBARGA1145−33.45054.88028.4621.0069.40C
ATOM2742CGARGA1145−32.21555.69728.1031.0072.46C
ATOM2743CDARGA1145−32.61956.97127.3681.0071.18C
ATOM2744NEARGA1145−31.44657.73226.9311.0074.68N
ATOM2745CZARGA1145−31.48858.84026.1861.0081.63C
ATOM2746NH1ARGA1145−32.66159.35325.7691.0076.56N
ATOM2747NH2ARGA1145−30.35459.45325.8441.0080.74N
ATOM2748NALAA1146−32.63254.10531.2721.0068.56N
ATOM2749CAALAA1146−31.78254.26532.4371.0070.12C
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ATOM2751OALAA1146−30.03952.81533.1941.0070.80O
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ATOM2753NLYSA1147−32.02951.86232.7861.0069.92N
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ATOM2755CLYSA1147−30.36550.17732.3221.0072.04C
ATOM2756OLYSA1147−29.31749.79432.8811.0066.93O
ATOM2757CBLYSA1147−32.71249.51432.9921.0073.45C
ATOM2758CGLYSA1147−32.53648.17733.6651.0078.51C
ATOM2759CDLYSA1147−33.83547.34833.4771.0082.18C
ATOM2760CELYSA1147−33.76245.95634.1031.0093.88C
ATOM2761NZLYSA1147−33.81645.96735.6171.0096.78N
ATOM2762NARGA1148−30.49450.32330.9861.0073.00N
ATOM2763CAARGA1148−29.39350.01630.0511.0070.89C
ATOM2764CARGA1148−28.13550.86030.2391.0074.78C
ATOM2765OARGA1148−27.03450.33430.0981.0080.02O
ATOM2766CBARGA1148−29.83250.15028.5901.0072.03C
ATOM2767CGARGA1148−30.78049.06828.0991.0070.33C
ATOM2768CDARGA1148−30.93649.10226.5961.0072.06C
ATOM2769NEARGA1148−31.67450.29026.1581.0071.65N
ATOM2770CZARGA1148−33.00650.39126.0841.0072.14C
ATOM2771NH1ARGA1148−33.80149.38226.4161.0078.11N
ATOM2772NH2ARGA1148−33.56851.52325.6711.0071.64N
ATOM2773NVALA1149−28.27052.15030.5471.0074.24N
ATOM2774CAVALA1149−27.07252.99030.7521.0071.90C
ATOM2775CVALA1149−26.39952.63232.0981.0074.21C
ATOM2776OVALA1149−25.18852.47832.1641.0077.27O
ATOM2777CBVALA1149−27.37454.51030.6761.0078.88C
ATOM2778CG1VALA1149−26.12255.31631.0061.0078.08C
ATOM2779CG2VALA1149−27.93954.92229.2821.0063.48C
ATOM2780NILEA1150−27.18252.49833.1581.0070.73N
ATOM2781CAILEA1150−26.62352.16434.4671.0070.91C
ATOM2782CILEA1150−25.90150.82334.4371.0069.25C
ATOM2783OILEA1150−24.74450.72734.8641.0074.32O
ATOM2784CBILEA1150−27.68952.14535.5701.0071.99C
ATOM2785CG1ILEA1150−28.26453.54135.7931.0069.99C
ATOM2786CG2ILEA1150−27.07751.64536.8481.0070.14C
ATOM2787CD1ILEA1150−29.55053.53936.6371.0076.32C
ATOM2788NTHRA1151−26.57849.79333.9381.0067.61N
ATOM2789CATHRA1151−25.95448.47633.7071.0067.20C
ATOM2790CTHRA1151−24.64848.60832.9201.0070.88C
ATOM2791OTHRA1151−23.68347.88733.1901.0074.26O
ATOM2792CBTHRA1151−26.87647.55832.9051.0068.20C
ATOM2793OG1THRA1151−28.15847.49133.5411.0072.09O
ATOM2794CG2THRA1151−26.27046.15832.7761.0064.27C
ATOM2795NTHRA1152−24.61949.52731.9411.0067.75N
ATOM2796CATHRA1152−23.39949.78831.1571.0070.23C
ATOM2797CTHRA1152−22.32250.41732.0421.0069.27C
ATOM2798OTHRA1152−21.14550.06831.9241.0071.67O
ATOM2799CBTHRA1152−23.67650.69629.9411.0066.28C
ATOM2800OG1THRA1152−24.68050.07629.1211.0070.58O
ATOM2801CG2THRA1152−22.39650.93929.1571.0069.86C
ATOM2802NPHEA1153−22.71951.33732.9131.0068.71N
ATOM2803CAPHEA1153−21.79151.79633.9901.0074.06C
ATOM2804CPHEA1153−21.34550.64934.8901.0070.59C
ATOM2805OPHEA1153−20.23250.66235.3891.0076.73O
ATOM2806CBPHEA1153−22.39152.89234.8721.0071.58C
ATOM2807CGPHEA1153−22.35954.27434.2581.0075.33C
ATOM2808CD1PHEA1153−21.18054.80733.7781.0076.55C
ATOM2809CD2PHEA1153−23.50855.03434.1701.0079.26C
ATOM2810CE1PHEA1153−21.14756.05933.2241.0073.15C
ATOM2811CE2PHEA1153−23.47956.28233.6221.0072.03C
ATOM2812CZPHEA1153−22.29656.79933.1471.0071.84C
ATOM2813NARGA1154−22.19849.65535.1081.0070.31N
ATOM2814CAARGA1154−21.80148.49035.9401.0072.56C
ATOM2815CARGA1154−20.77447.55935.3121.0074.07C
ATOM2816OARGA1154−19.82647.15835.9941.0075.46O
ATOM2817CBARGA1154−23.01347.66136.3541.0071.60C
ATOM2818CGARGA1154−23.92648.39237.3151.0078.93C
ATOM2819CDARGA1154−24.31347.48438.4851.0080.49C
ATOM2820NEARGA1154−25.31246.49238.1261.0081.98N
ATOM2821CZARGA1154−25.65745.43638.8701.0084.48C
ATOM2822NH1ARGA1154−25.08245.18640.0631.0090.99N
ATOM2823NH2ARGA1154−26.59544.60138.4211.0088.13N
ATOM2824NTHRA1155−20.95647.21934.0291.0073.12N
ATOM2825CATHRA1155−20.09546.23233.3461.0074.07C
ATOM2826CTHRA1155−18.96946.82832.5011.0076.10C
ATOM2827OTHRA1155−17.91246.21832.3611.0076.98O
ATOM2828CBTHRA1155−20.94045.32432.4221.0075.54C
ATOM2829OG1THRA1155−21.36846.06331.2511.0076.27O
ATOM2830CG2THRA1155−22.15244.77733.1761.0073.31C
ATOM2831NGLYA1156−19.18548.00831.9361.0075.88N
ATOM2832CAGLYA1156−18.19848.59131.0381.0076.99C
ATOM2833CGLYA1156−18.11347.82929.7031.0078.89C
ATOM2834OGLYA1156−17.05447.78529.0731.0077.75O
ATOM2835NTHRA1157−19.24047.23529.2971.0079.45N
ATOM2836CATHRA1157−19.38846.57328.0251.0075.30C
ATOM2837CTHRA1157−20.71347.00727.4351.0076.85C
ATOM2838OTHRA1157−21.56847.59128.1211.0072.71O
ATOM2839CBTHRA1157−19.49145.06928.1501.0077.35C
ATOM2840OG1THRA1157−20.77144.74728.7181.0078.06O
ATOM2841CG2THRA1157−18.33244.46029.0161.0073.49C
ATOM2842NTRPA1158−20.88046.70626.1491.0075.57N
ATOM2843CATRPA1158−22.10247.00025.4121.0072.41C
ATOM2844CTRPA1158−23.21345.95625.5791.0072.74C
ATOM2845OTRPA1158−24.24346.07024.9041.0074.46O
ATOM2846CBTRPA1158−21.76947.10123.9271.0076.07C
ATOM2847CGTRPA1158−20.84448.14723.6461.0074.03C
ATOM2848CD1TRPA1158−19.56348.00723.2741.0071.12C
ATOM2849CD2TRPA1158−21.10749.53923.7171.0072.83C
ATOM2850NE1TRPA1158−18.98649.23823.0961.0076.80N
ATOM2851CE2TRPA1158−19.91650.20023.3621.0066.00C
ATOM2852CE3TRPA1158−22.23850.29724.0491.0074.60C
ATOM2853CZ2TRPA1158−19.81151.58223.3221.0071.92C
ATOM2854CZ3TRPA1158−22.13851.67324.0121.0076.31C
ATOM2855CH2TRPA1158−20.92752.30723.6491.0074.45C
ATOM2856NASPA1159−23.01944.95526.4601.0072.02N
ATOM2857CAASPA1159−23.96743.83526.6641.0071.28C
ATOM2858CASPA1159−25.45044.20726.8371.0072.00C
ATOM2859OASPA1159−26.31643.42226.4501.0073.98O
ATOM2860CBASPA1159−23.52742.96627.8521.0073.56C
ATOM2861CGASPA1159−22.25442.14327.5701.0078.31C
ATOM2862OD1ASPA1159−21.71542.17826.4501.0078.77O
ATOM2863OD2ASPA1159−21.79141.44928.4991.0092.78O
ATOM2864NALAA1160−25.74645.38127.4051.0072.33N
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ATOM2866CALAA1160−27.81546.21526.2381.0072.65C
ATOM2867OALAA1160−29.03746.28526.1691.0075.90O
ATOM2868CBALAA1160−27.25446.99828.5601.0069.43C
ATOM2869NTYRA1161−27.01046.46525.2001.0073.56N
ATOM2870CATYRA1161−27.50046.85023.8701.0070.85C
ATOM2871CTYRA1161−27.50045.69322.8451.0071.12C
ATOM2872OTYRA1161−27.77945.92821.6841.0074.87O
ATOM2873CBTYRA1161−26.71248.06723.3811.0070.71C
ATOM2874CGTYRA1161−26.97749.22424.2821.0069.57C
ATOM2875CD1TYRA1161−28.02050.09024.0371.0067.53C
ATOM2876CD2TYRA1161−26.18749.45125.4001.0071.83C
ATOM2877CE1TYRA1161−28.26951.15724.8781.0069.80C
ATOM2878CE2TYRA1161−26.43250.50926.2361.0064.36C
ATOM2879CZTYRA1161−27.47051.36625.9801.0073.12C
ATOM2880OHTYRA1161−27.69552.43626.8501.0077.23O
ATOM2881NLYSA263−27.19344.46823.2891.0068.62N
ATOM2882CALYSA263−27.21143.30222.4371.0068.39C
ATOM2883CLYSA263−28.61742.99222.0391.0068.68C
ATOM2884OLYSA263−29.49743.03222.8731.0071.28O
ATOM2885CBLYSA263−26.71642.05223.1521.0070.24C
ATOM2886CGLYSA263−25.24741.95723.3911.0076.63C
ATOM2887CDLYSA263−24.91640.56723.9661.0077.14C
ATOM2888CELYSA263−23.43340.25723.9261.0086.04C
ATOM2889NZLYSA263−23.13938.78124.0831.0088.05N
ATOM2890NPHEA264−28.82342.68320.7661.0066.31N
ATOM2891CAPHEA264−30.12942.26720.2401.0067.14C
ATOM2892CPHEA264−31.24743.27220.4991.0066.08C
ATOM2893OPHEA264−32.38542.89220.7541.0066.16O
ATOM2894CBPHEA264−30.49040.87720.7741.0065.00C
ATOM2895CGPHEA264−29.45539.84320.4611.0068.77C
ATOM2896CD1PHEA264−28.76139.18221.4651.0069.39C
ATOM2897CD2PHEA264−29.16639.53019.1351.0069.77C
ATOM2898CE1PHEA264−27.80038.22321.1481.0067.15C
ATOM2899CE2PHEA264−28.21038.57818.8251.0068.88C
ATOM2900CZPHEA264−27.53137.92519.8321.0067.43C
ATOM2901NCYSA265−30.89144.55120.4301.0066.76N
ATOM2902CACYSA265−31.83745.63620.4941.0066.33C
ATOM2903CCYSA265−32.19246.03419.0511.0066.21C
ATOM2904OCYSA265−33.35546.21718.7421.0067.25O
ATOM2905CBCYSA265−31.26946.81821.2741.0065.09C
ATOM2906SGCYSA265−31.06646.52123.1101.0069.25S
ATOM2907NLEUA266−31.18346.16318.1861.0063.62N
ATOM2908CALEUA266−31.38546.56016.8061.0065.11C
ATOM2909CLEUA266−32.13045.48716.0371.0065.91C
ATOM2910OLEUA266−31.81544.31216.1721.0065.41O
ATOM2911CBLEUA266−30.04446.78716.1161.0063.47C
ATOM2912CGLEUA266−29.13747.92816.5671.0066.69C
ATOM2913CD1LEUA266−27.79147.82715.8481.0064.50C
ATOM2914CD2LEUA266−29.79649.27116.3251.0066.85C
ATOM2915NLYSA267−33.11245.89815.2381.0068.75N
ATOM2916CALYSA267−33.87044.97314.3501.0071.90C
ATOM2917CLYSA267−32.97344.00713.5661.0069.09C
ATOM2918OLYSA267−33.27442.82913.4631.0072.39O
ATOM2919CBLYSA267−34.76845.76213.3731.0074.97C
ATOM2920CGLYSA267−34.00846.72112.4191.0082.81C
ATOM2921CDLYSA267−34.91347.65611.6341.0084.17C
ATOM2922CELYSA267−34.07148.65810.8161.0088.72C
ATOM2923NZLYSA267−34.90449.77710.2901.0088.47N
ATOM2924NGLUA268−31.87744.51913.0241.0068.14N
ATOM2925CAGLUA268−30.92943.70612.2571.0068.69C
ATOM2926CGLUA268−30.23242.66513.1251.0069.97C
ATOM2927OGLUA268−30.01041.55412.6791.0071.10O
ATOM2928CBGLUA268−29.89744.56811.4921.0071.25C
ATOM2929CGGLUA268−29.06345.59112.3181.0082.60C
ATOM2930CDGLUA268−29.72046.97512.4911.0084.41C
ATOM2931OE1GLUA268−30.93447.16412.2191.0081.45O
ATOM2932OE2GLUA268−28.99247.87712.9111.0083.78O
ATOM2933NHISA269−29.89143.02314.3661.0070.32N
ATOM2934CAHISA269−29.27442.06515.2871.0068.22C
ATOM2935CHISA269−30.29041.03915.7571.0066.82C
ATOM2936OHISA269−29.94639.85615.8821.0065.02O
ATOM2937CBHISA269−28.61242.79416.4511.0071.92C
ATOM2938CGHISA269−27.39043.55416.0471.0071.60C
ATOM2939ND1HISA269−26.54744.13416.9581.0068.24N
ATOM2940CD2HISA269−26.86743.82414.8241.0075.13C
ATOM2941CE1HISA269−25.55744.73416.3251.0073.61C
ATOM2942NE2HISA269−25.72544.56215.0271.0078.78N
ATOM2943NLYSA270−31.52641.47716.0141.0064.48N
ATOM2944CALYSA270−32.61940.53616.2961.0067.98C
ATOM2945CLYSA270−32.76139.52115.1461.0068.82C
ATOM2946OLYSA270−32.86138.32815.3941.0066.36O
ATOM2947CBLYSA270−33.94841.24716.5191.0066.79C
ATOM2948CGLYSA270−33.98742.12717.7731.0073.23C
ATOM2949CDLYSA270−35.41242.55618.0881.0071.51C
ATOM2950CELYSA270−35.48743.40519.3451.0075.87C
ATOM2951NZLYSA270−35.01742.66920.5451.0077.55N
ATOM2952NALAA271−32.76440.00913.8971.0069.99N
ATOM2953CAALAA271−32.85539.12112.7131.0071.38C
ATOM2954CALAA271−31.73138.11112.7271.0071.12C
ATOM2955OALAA271−31.97536.94612.4661.0073.66O
ATOM2956CBALAA271−32.85539.91811.3991.0064.88C
ATOM2957NLEUA272−30.50238.54813.0341.0072.07N
ATOM2958CALEUA272−29.35937.59913.1511.0074.58C
ATOM2959CLEUA272−29.56136.51314.2401.0075.85C
ATOM2960OLEUA272−29.13735.36714.0511.0081.93O
ATOM2961CBLEUA272−28.02538.32413.4081.0073.91C
ATOM2962CGLEUA272−27.40639.22812.3291.0078.65C
ATOM2963CD1LEUA272−26.12939.86412.8731.0079.09C
ATOM2964CD2LEUA272−27.11838.46511.0801.0065.55C
ATOM2965NLYSA273−30.19536.86415.3581.0076.42N
ATOM2966CALYSA273−30.50935.86616.4031.0077.14C
ATOM2967CLYSA273−31.51234.84515.8571.0077.91C
ATOM2968OLYSA273−31.34633.66316.0831.0076.67O
ATOM2969CBLYSA273−31.04936.52917.6561.0078.11C
ATOM2970CGLYSA273−31.22635.60818.8671.0080.19C
ATOM2971CDLYSA273−31.47336.44320.1001.0079.05C
ATOM2972CELYSA273−31.75235.62421.3291.0082.30C
ATOM2973NZLYSA273−31.95336.54422.4921.0084.12N
ATOM2974NTHRA274−32.54135.31315.1391.0080.30N
ATOM2975CATHRA274−33.52534.41414.4871.0078.23C
ATOM2976CTHRA274−32.82733.36913.6511.0076.51C
ATOM2977OTHRA274−33.08232.19113.8261.0075.03O
ATOM2978CBTHRA274−34.52035.16613.5641.0081.00C
ATOM2979OG1THRA274−35.19836.19714.2931.0083.35O
ATOM2980CG2THRA274−35.55334.20612.9631.0079.62C
ATOM2981NLEUA275−31.95033.80012.7541.0073.92N
ATOM2982CALEUA275−31.18332.86211.9251.0074.39C
ATOM2983CLEUA275−30.33031.92712.7791.0075.45C
ATOM2984OLEUA275−30.27930.72112.5241.0076.13O
ATOM2985CBLEUA275−30.30133.60510.9221.0075.70C
ATOM2986CGLEUA275−30.97534.4179.8081.0075.16C
ATOM2987CD1LEUA275−29.90935.1698.9811.0067.26C
ATOM2988CD2LEUA275−31.84633.5338.9101.0070.95C
ATOM2989NGLYA276−29.66332.47613.7971.0078.78N
ATOM2990CAGLYA276−28.89031.66114.7401.0073.44C
ATOM2991CGLYA276−29.74030.60515.4221.0073.61C
ATOM2992OGLYA276−29.24729.51515.7431.0074.08O
ATOM2993NILEA277−31.01730.92115.6451.0073.26N
ATOM2994CAILEA277−31.95529.98716.2861.0076.95C
ATOM2995CILEA277−32.35428.86215.3341.0075.85C
ATOM2996OILEA277−32.54027.72015.7681.0077.29O
ATOM2997CBILEA277−33.20230.72116.8661.0074.67C
ATOM2998CG1ILEA277−32.79731.55818.0811.0076.85C
ATOM2999CG2ILEA277−34.27529.74717.2871.0072.00C
ATOM3000CD1ILEA277−33.97032.23918.7881.0076.44C
ATOM3001NILEA278−32.48129.18814.0481.0079.83N
ATOM3002CAILEA278−32.78728.20813.0101.0077.27C
ATOM3003CILEA278−31.63027.22512.9311.0079.77C
ATOM3004OILEA278−31.83326.00412.8771.0080.80O
ATOM3005CBILEA278−33.00428.89211.6371.0077.96C
ATOM3006CG1ILEA278−34.24429.79811.6511.0079.43C
ATOM3007CG2ILEA278−33.17427.88810.5571.0078.57C
ATOM3008CD1ILEA278−35.54429.08711.7751.0083.47C
ATOM3009NMETA279−30.41227.76112.9331.0079.21N
ATOM3010CAMETA279−29.20626.94212.8261.0077.35C
ATOM3011CMETA279−29.01926.04414.0181.0075.85C
ATOM3012OMETA279−28.79824.84413.8521.0073.53O
ATOM3013CBMETA279−27.98227.83012.6321.0076.42C
ATOM3014CGMETA279−27.99228.53511.2841.0079.03C
ATOM3015SDMETA279−26.85229.89711.1561.0080.66S
ATOM3016CEMETA279−27.06130.3049.4071.0079.86C
ATOM3017NGLYA280−29.11226.62515.2141.0078.82N
ATOM3018CAGLYA280−28.93725.88016.4631.0076.24C
ATOM3019CGLYA280−29.97024.78516.6601.0079.31C
ATOM3020OGLYA280−29.64023.69417.1201.0080.01O
ATOM3021NTHRA281−31.21925.08416.3091.0080.59N
ATOM3022CATHRA281−32.30924.12316.4081.0078.83C
ATOM3023CTHRA281−32.05722.93515.4921.0077.28C
ATOM3024OTHRA281−32.19021.80115.9231.0076.07O
ATOM3025CBTHRA281−33.65724.78016.0701.0081.71C
ATOM3026OG1THRA281−33.96425.77117.0651.0079.40O
ATOM3027CG2THRA281−34.76923.76016.0381.0082.36C
ATOM3028NPHEA282−31.69423.20514.2371.0079.67N
ATOM3029CAPHEA282−31.34722.14713.2791.0077.00C
ATOM3030CPHEA282−30.22121.28213.8171.0076.25C
ATOM3031OPHEA282−30.26620.06913.6971.0073.80O
ATOM3032CBPHEA282−30.91722.73111.9301.0077.95C
ATOM3033CGPHEA282−30.69121.67910.8531.0076.73C
ATOM3034CD1PHEA282−31.72321.3149.9951.0075.49C
ATOM3035CD2PHEA282−29.46021.06410.7061.0076.37C
ATOM3036CE1PHEA282−31.52620.3438.9971.0076.36C
ATOM3037CE2PHEA282−29.25420.0979.7201.0084.43C
ATOM3038CZPHEA282−30.29719.7378.8571.0076.69C
ATOM3039NTHRA283−29.21421.92314.4061.0077.11N
ATOM3040CATHRA283−28.05821.21814.9451.0076.40C
ATOM3041CTHRA283−28.46020.31116.0781.0076.62C
ATOM3042OTHRA283−28.10519.15216.0731.0076.69O
ATOM3043CBTHRA283−26.98122.18315.4481.0076.06C
ATOM3044OG1THRA283−26.54423.02314.3671.0080.64O
ATOM3045CG2THRA283−25.78421.41216.0181.0075.59C
ATOM3046NLEUA284−29.20320.84417.0411.0078.59N
ATOM3047CALEUA284−29.63620.06518.2041.0077.27C
ATOM3048CLEUA284−30.57818.92017.8451.0077.57C
ATOM3049OLEUA284−30.56017.88618.5001.0076.25O
ATOM3050CBLEUA284−30.30020.98619.2361.0080.83C
ATOM3051CGLEUA284−29.35922.01319.9161.0089.13C
ATOM3052CD1LEUA284−30.16223.02920.7241.0093.20C
ATOM3053CD2LEUA284−28.29021.33720.8011.0091.45C
ATOM3054NCYSA285−31.39519.10816.8061.0077.86N
ATOM3055CACYSA285−32.34118.07316.3671.0076.99C
ATOM3056CCYSA285−31.67716.92015.6201.0075.92C
ATOM3057OCYSA285−32.09515.78215.7831.0075.32O
ATOM3058CBCYSA285−33.46218.67815.5131.0076.74C
ATOM3059SGCYSA285−34.72719.54116.5111.0086.35S
ATOM3060NTRPA286−30.65717.21914.8101.0076.29N
ATOM3061CATRPA286−29.93816.19214.0201.0075.01C
ATOM3062CTRPA286−28.63515.67114.6351.0075.15C
ATOM3063OTRPA286−28.25014.54414.3301.0075.31O
ATOM3064CBTRPA286−29.64116.70312.5991.0072.75C
ATOM3065CGTRPA286−30.85316.73811.7661.0070.37C
ATOM3066CD1TRPA286−31.54317.83211.3811.0069.67C
ATOM3067CD2TRPA286−31.53315.61311.2121.0067.81C
ATOM3068NE1TRPA286−32.62117.46910.6141.0076.08N
ATOM3069CE2TRPA286−32.63616.10710.4961.0067.36C
ATOM3070CE3TRPA286−31.31414.22811.2541.0069.38C
ATOM3071CZ2TRPA286−33.52315.2749.8221.0073.52C
ATOM3072CZ3TRPA286−32.20213.39110.5811.0071.27C
ATOM3073CH2TRPA286−33.29113.9209.8751.0072.45C
ATOM3074NLEUA287−27.95816.45415.4741.0071.19N
ATOM3075CALEUA287−26.64516.02016.0011.0072.17C
ATOM3076CLEUA287−26.66914.65116.6671.0070.61C
ATOM3077OLEUA287−25.82213.83416.3471.0073.46O
ATOM3078CBLEUA287−26.01917.04916.9531.0074.35C
ATOM3079CGLEUA287−24.54216.88417.2771.0076.88C
ATOM3080CD1LEUA287−23.68216.96416.0131.0078.66C
ATOM3081CD2LEUA287−24.13017.95618.2791.0079.06C
ATOM3082NPROA288−27.62714.39517.5831.0069.58N
ATOM3083CAPROA288−27.68213.07418.2271.0068.99C
ATOM3084CPROA288−27.76711.90317.2481.0068.90C
ATOM3085OPROA288−27.05210.90617.4061.0067.39O
ATOM3086CBPROA288−28.97913.14019.0571.0068.33C
ATOM3087CGPROA288−29.20714.54619.3051.0066.28C
ATOM3088CDPROA288−28.70115.26918.0911.0070.69C
ATOM3089NPHEA289−28.64112.04216.2551.0067.58N
ATOM3090CAPHEA289−28.80711.03815.2171.0067.91C
ATOM3091CPHEA289−27.47910.74614.5191.0069.22C
ATOM3092OPHEA289−27.0729.57614.4121.0068.30O
ATOM3093CBPHEA289−29.85311.50514.1891.0069.82C
ATOM3094CGPHEA289−30.01410.57813.0471.0067.38C
ATOM3095CD1PHEA289−30.8509.47513.1571.0064.32C
ATOM3096CD2PHEA289−29.33510.79511.8591.0071.12C
ATOM3097CE1PHEA289−31.0088.61012.1141.0070.14C
ATOM3098CE2PHEA289−29.4899.92610.7981.0076.34C
ATOM3099CZPHEA289−30.3278.82910.9231.0075.79C
ATOM3100NPHEA290−26.81811.81114.0521.0069.03N
ATOM3101CAPHEA290−25.52211.68613.3471.0070.29C
ATOM3102CPHEA290−24.34611.25414.2221.0070.41C
ATOM3103OPHEA290−23.38610.67713.6971.0072.74O
ATOM3104CBPHEA290−25.18012.97112.5851.0073.06C
ATOM3105CGPHEA290−25.94213.10811.2951.0073.80C
ATOM3106CD1PHEA290−27.10313.86411.2261.0070.64C
ATOM3107CD2PHEA290−25.49412.46710.1491.0076.58C
ATOM3108CE1PHEA290−27.80013.99010.0581.0079.74C
ATOM3109CE2PHEA290−26.19112.5868.9601.0075.30C
ATOM3110CZPHEA290−27.34913.3528.9141.0077.36C
ATOM3111NILEA291−24.40411.52215.5281.0069.93N
ATOM3112CAILEA291−23.39111.00316.4541.0071.11C
ATOM3113CILEA291−23.5179.47616.5161.0072.55C
ATOM3114OILEA291−22.5078.76516.5341.0076.20O
ATOM3115CBILEA291−23.51111.62117.8731.0071.52C
ATOM3116CG1ILEA291−23.07313.08117.8531.0073.15C
ATOM3117CG2ILEA291−22.64210.88818.8941.0065.02C
ATOM3118CD1ILEA291−23.30913.80719.1401.0072.16C
ATOM3119NVALA292−24.7578.98416.5451.0074.02N
ATOM3120CAVALA292−25.0237.53916.6191.0074.95C
ATOM3121CVALA292−24.5866.78215.3401.0073.29C
ATOM3122OVALA292−24.1925.61915.4261.0074.16O
ATOM3123CBVALA292−26.5057.25316.9981.0074.89C
ATOM3124CG1VALA292−26.8145.77516.9151.0073.02C
ATOM3125CG2VALA292−26.7817.77418.4271.0073.75C
ATOM3126NASNA293−24.6517.43114.1761.0073.79N
ATOM3127CAASNA293−24.1126.83512.9361.0072.79C
ATOM3128CASNA293−22.6416.49513.0851.0071.66C
ATOM3129OASNA293−22.2065.40912.7091.0072.11O
ATOM3130CBASNA293−24.2507.78211.7451.0072.08C
ATOM3131CGASNA293−25.6737.96111.2871.0076.70C
ATOM3132OD1ASNA293−26.5847.30611.7771.0077.56O
ATOM3133ND2ASNA293−25.8718.86310.3271.0072.62N
ATOM3134NILEA294−21.8877.43513.6391.0070.24N
ATOM3135CAILEA294−20.4517.27213.8331.0071.74C
ATOM3136CILEA294−20.1566.29614.9631.0071.03C
ATOM3137OILEA294−19.2885.43714.8071.0069.26O
ATOM3138CBILEA294−19.7848.60814.1151.0071.93C
ATOM3139CG1ILEA294−19.8789.50212.8851.0071.27C
ATOM3140CG2ILEA294−18.3138.42114.4981.0072.08C
ATOM3141CD1ILEA294−19.39110.86213.1681.0079.13C
ATOM3142NVALA295−20.8726.42616.0881.0071.55N
ATOM3143CAVALA295−20.7355.48417.2341.0070.36C
ATOM3144CVALA295−20.9324.02916.7821.0069.42C
ATOM3145OVALA295−20.2243.14217.2431.0066.44O
ATOM3146CBVALA295−21.7205.83518.3961.0070.22C
ATOM3147CG1VALA295−21.8034.72119.4191.0068.33C
ATOM3148CG2VALA295−21.3047.12519.0651.0071.01C
ATOM3149NHISA296−21.8903.80515.8781.0069.19N
ATOM3150CAHISA296−22.1282.46215.3111.0071.67C
ATOM3151CHISA296−21.0791.97714.2971.0072.30C
ATOM3152OHISA296−21.0220.78513.9961.0072.47O
ATOM3153CBHISA296−23.5452.34914.7371.0072.70C
ATOM3154CGHISA296−24.5912.26915.7951.0077.53C
ATOM3155ND1HISA296−24.3691.63216.9911.0078.84N
ATOM3156CD2HISA296−25.8592.73615.8481.0085.05C
ATOM3157CE1HISA296−25.4481.71017.7331.0078.27C
ATOM3158NE2HISA296−26.3702.37317.0691.0081.35N
ATOM3159NVALA297−20.2672.89313.7811.0073.43N
ATOM3160CAVALA297−19.1182.53512.9481.0073.61C
ATOM3161CVALA297−17.9932.05113.8781.0072.62C
ATOM3162OVALA297−17.2201.16513.5051.0073.05O
ATOM3163CBVALA297−18.6533.71312.0501.0073.85C
ATOM3164CG1VALA297−17.4243.33811.2381.0072.56C
ATOM3165CG2VALA297−19.7864.13911.1271.0072.80C
ATOM3166NILEA298−17.9122.63615.0821.0072.35N
ATOM3167CAILEA298−16.9302.23616.0971.0073.03C
ATOM3168CILEA298−17.3510.88616.6881.0073.53C
ATOM3169OILEA298−16.612−0.09916.5971.0074.08O
ATOM3170CBILEA298−16.7803.28817.2461.0073.65C
ATOM3171CG1ILEA298−16.4024.68716.7161.0076.60C
ATOM3172CG2ILEA298−15.7572.82718.2551.0073.25C
ATOM3173CD1ILEA298−15.0594.76416.0071.0080.21C
ATOM3174NGLNA299−18.5420.86417.2901.0073.48N
ATOM3175CAGLNA299−19.132−0.34517.8621.0074.55C
ATOM3176CGLNA299−20.619−0.35417.5241.0075.17C
ATOM3177OGLNA299−21.3680.46618.0401.0077.55O
ATOM3178CBGLNA299−18.917−0.40119.3781.0075.38C
ATOM3179CGGLNA299−19.532−1.63020.0861.0076.95C
ATOM3180CDGLNA299−18.993−2.96319.5631.0080.36C
ATOM3181OE1GLNA299−17.801−3.25519.6751.0081.59O
ATOM3182NE2GLNA299−19.883−3.77818.9921.0079.18N
ATOM3183NASPA300−21.035−1.28216.6621.0075.16N
ATOM3184CAASPA300−22.420−1.35716.2061.0076.26C
ATOM3185CASPA300−23.306−1.99417.2841.0076.70C
ATOM3186OASPA300−22.818−2.75618.1211.0075.98O
ATOM3187CBASPA300−22.492−2.14514.8831.0076.84C
ATOM3188CGASPA300−23.781−1.87314.0691.0078.37C
ATOM3189OD1ASPA300−24.504−0.87414.3031.0080.03O
ATOM3190OD2ASPA300−24.068−2.68813.1681.0083.02O
ATOM3191NASNA301−24.601−1.66117.2481.0076.86N
ATOM3192CAASNA301−25.633−2.19618.1771.0076.17C
ATOM3193CASNA301−25.536−1.74019.6561.0075.65C
ATOM3194OASNA301−26.220−2.29020.5201.0076.59O
ATOM3195CBASNA301−25.700−3.74118.0891.0075.63C
ATOM3196CGASNA301−26.022−4.23516.6901.0074.71C
ATOM3197OD1ASNA301−26.917−3.71416.0301.0064.90O
ATOM3198ND2ASNA301−25.291−5.24616.2331.0075.78N
ATOM3199NLEUA302−24.700−0.74319.9381.0076.35N
ATOM3200CALEUA302−24.547−0.19721.2981.0078.80C
ATOM3201CLEUA302−25.8150.53421.8171.0079.26C
ATOM3202OLEUA302−26.0740.56623.0181.0080.59O
ATOM3203CBLEUA302−23.3500.76421.3351.0079.16C
ATOM3204CGLEUA302−22.8321.21322.7031.0079.37C
ATOM3205CD1LEUA302−22.1990.03923.4381.0077.36C
ATOM3206CD2LEUA302−21.8392.35122.5451.0079.97C
ATOM3207NILEA303−26.5721.10520.8881.0079.64N
ATOM3208CAILEA303−27.7651.88621.1201.0080.47C
ATOM3209CILEA303−28.8811.16620.3931.0080.12C
ATOM3210OILEA303−28.7470.85419.2081.0080.56O
ATOM3211CBILEA303−27.6233.31320.5731.0079.71C
ATOM3212CG1ILEA303−26.5134.05621.3261.0080.55C
ATOM3213CG2ILEA303−28.9304.06820.7081.0078.77C
ATOM3214CD1ILEA303−26.2435.42720.8261.0083.25C
ATOM3215NARGA304−29.9730.91021.1111.0081.10N
ATOM3216CAARGA304−31.1320.17720.5961.0083.66C
ATOM3217CARGA304−31.6860.77619.3101.0082.48C
ATOM3218OARGA304−31.5421.97719.0701.0083.57O
ATOM3219CBARGA304−32.2650.19221.6081.0084.33C
ATOM3220CGARGA304−31.969−0.42622.9661.0091.17C
ATOM3221CDARGA304−33.142−0.22723.8701.0096.90C
ATOM3222NEARGA304−34.321−0.91423.3471.00107.82N
ATOM3223CZARGA304−35.556−0.82023.8451.00114.88C
ATOM3224NH1ARGA304−35.826−0.05624.9091.00119.98N
ATOM3225NH2ARGA304−36.539−1.50523.2681.00117.19N
ATOM3226NLYSA305−32.317−0.06918.4961.0080.96N
ATOM3227CALYSA305−32.9500.36917.2491.0082.83C
ATOM3228CLYSA305−34.0241.43217.5011.0082.19C
ATOM3229OLYSA305−34.1222.39916.7511.0083.88O
ATOM3230CBLYSA305−33.573−0.81916.5141.0082.92C
ATOM3231CGLYSA305−34.091−0.49915.1091.0086.35C
ATOM3232CDLYSA305−34.754−1.70914.4721.0087.13C
ATOM3233CELYSA305−35.417−1.32913.1541.0088.05C
ATOM3234NZLYSA305−36.147−2.48112.5521.0091.16N
ATOM3235NGLUA306−34.8131.23718.5531.0079.77N
ATOM3236CAGLUA306−35.8882.16618.9281.0078.91C
ATOM3237CGLUA306−35.3883.53419.3541.0075.42C
ATOM3238OGLUA306−36.0834.52619.1561.0073.51O
ATOM3239CBGLUA306−36.7071.58620.0621.0080.27C
ATOM3240CGGLUA306−37.4640.32319.6821.0088.15C
ATOM3241CDGLUA306−38.035−0.37920.8761.0092.42C
ATOM3242OE1GLUA306−38.0870.24521.9641.00103.55O
ATOM3243OE2GLUA306−38.436−1.56020.7401.00104.10O
ATOM3244NVALA307−34.1913.58619.9381.0074.31N
ATOM3245CAVALA307−33.5794.86420.3141.0073.09C
ATOM3246CVALA307−33.1265.56319.0401.0072.26C
ATOM3247OVALA307−33.4156.74018.8541.0074.87O
ATOM3248CBVALA307−32.3984.70321.3301.0071.64C
ATOM3249CG1VALA307−31.5975.98621.4411.0063.03C
ATOM3250CG2VALA307−32.9254.28722.7031.0068.24C
ATOM3251NTYRA308−32.4234.83318.1771.0071.72N
ATOM3252CATYRA308−31.9555.36616.8921.0072.58C
ATOM3253CTYRA308−33.1065.89216.0251.0072.20C
ATOM3254OTYRA308−32.9816.95115.4141.0072.72O
ATOM3255CBTYRA308−31.1844.29416.1351.0075.16C
ATOM3256CGTYRA308−30.5344.77014.8551.0075.61C
ATOM3257CD1TYRA308−30.9844.33813.6101.0083.25C
ATOM3258CD2TYRA308−29.4705.65014.8941.0077.87C
ATOM3259CE1TYRA308−30.3784.78012.4351.0082.87C
ATOM3260CE2TYRA308−28.8596.09813.7471.0074.78C
ATOM3261CZTYRA308−29.3135.66312.5131.0080.83C
ATOM3262OHTYRA308−28.7166.10011.3571.0079.13O
ATOM3263NILEA309−34.2145.15215.9791.0070.18N
ATOM3264CAILEA309−35.4125.59315.2511.0071.74C
ATOM3265CILEA309−35.9596.87315.8631.0072.46C
ATOM3266OILEA309−36.2727.79815.1391.0073.87O
ATOM3267CBILEA309−36.5194.52015.2431.0070.27C
ATOM3268CG1ILEA309−36.1413.37514.3161.0069.96C
ATOM3269CG2ILEA309−37.8395.10014.7811.0068.79C
ATOM3270CD1ILEA309−36.9872.14514.5161.0072.32C
ATOM3271NLEUA310−36.0676.91717.1951.0074.30N
ATOM3272CALEUA310−36.5618.10917.8951.0073.71C
ATOM3273CLEUA310−35.6829.32417.6191.0075.46C
ATOM3274OLEUA310−36.20810.41617.4141.0076.09O
ATOM3275CBLEUA310−36.6777.87319.4051.0076.16C
ATOM3276CGLEUA310−37.1039.08720.2691.0079.17C
ATOM3277CD1LEUA310−38.3539.78719.6991.0080.03C
ATOM3278CD2LEUA310−37.3448.66621.7101.0074.92C
ATOM3279NLEUA311−34.3589.14217.6121.0074.63N
ATOM3280CALEUA311−33.43610.24917.2711.0073.90C
ATOM3281CLEUA311−33.61110.74415.8291.0073.58C
ATOM3282OLEUA311−33.34611.90415.5461.0075.24O
ATOM3283CBLEUA311−31.9769.85717.4971.0071.95C
ATOM3284CGLEUA311−31.5839.46818.9261.0074.42C
ATOM3285CD1LEUA311−30.0949.20218.9691.0078.05C
ATOM3286CD2LEUA311−31.96010.52519.9511.0079.68C
ATOM3287NASNA312−34.0539.86214.9321.0073.28N
ATOM3288CAASNA312−34.33710.22813.5511.0074.59C
ATOM3289CASNA312−35.60211.07613.4911.0074.96C
ATOM3290OASNA312−35.65412.05712.7411.0076.71O
ATOM3291CBASNA312−34.4908.97012.6791.0073.89C
ATOM3292CGASNA312−34.2769.23111.1701.0075.40C
ATOM3293OD1ASNA312−33.9338.30210.4391.0075.69O
ATOM3294ND2ASNA312−34.47110.47110.7091.0073.01N
ATOM3295NTRPA313−36.61410.70214.2751.0075.65N
ATOM3296CATRPA313−37.88011.46114.3271.0073.90C
ATOM3297CTRPA313−37.74812.84414.9671.0074.02C
ATOM3298OTRPA313−38.55613.72614.6761.0074.92O
ATOM3299CBTRPA313−38.98810.63414.9861.0075.64C
ATOM3300CGTRPA313−39.5629.76813.9841.0077.34C
ATOM3301CD1TRPA313−39.0708.58313.5481.0077.95C
ATOM3302CD2TRPA313−40.75710.00413.2541.0077.66C
ATOM3303NE1TRPA313−39.8858.05612.5891.0077.47N
ATOM3304CE2TRPA313−40.9358.91012.3841.0079.44C
ATOM3305CE3TRPA313−41.70111.03713.2471.0076.72C
ATOM3306CZ2TRPA313−42.0258.81311.5091.0079.40C
ATOM3307CZ3TRPA313−42.79010.94412.3741.0078.33C
ATOM3308CH2TRPA313−42.9409.83511.5181.0077.48C
ATOM3309NILEA314−36.74213.02415.8221.0071.91N
ATOM3310CAILEA314−36.41114.33616.3681.0071.31C
ATOM3311CILEA314−35.84715.18515.2131.0071.78C
ATOM3312OILEA314−36.10816.39615.1141.0072.42O
ATOM3313CBILEA314−35.40614.23917.5461.0069.93C
ATOM3314CG1ILEA314−36.04013.51918.7351.0069.22C
ATOM3315CG2ILEA314−34.97515.61118.0021.0066.77C
ATOM3316CD1ILEA314−35.05413.19519.8411.0070.69C
ATOM3317NGLYA315−35.07214.53614.3501.0071.45N
ATOM3318CAGLYA315−34.60315.15013.1181.0073.83C
ATOM3319CGLYA315−35.78015.52012.2371.0073.35C
ATOM3320OGLYA315−35.91316.67311.8661.0071.64O
ATOM3321NTYRA316−36.63414.54511.9091.0074.36N
ATOM3322CATYRA316−37.82514.80011.0501.0074.23C
ATOM3323CTYRA316−38.66815.97011.5261.0075.48C
ATOM3324OTYRA316−38.94916.88910.7541.0076.19O
ATOM3325CBTYRA316−38.74613.57010.9601.0074.65C
ATOM3326CGTYRA316−38.25612.35910.1391.0077.26C
ATOM3327CD1TYRA316−38.86911.12110.2881.0077.77C
ATOM3328CD2TYRA316−37.20012.4489.2311.0075.43C
ATOM3329CE1TYRA316−38.45910.0259.5711.0071.53C
ATOM3330CE2TYRA316−36.78711.3588.5141.0078.76C
ATOM3331CZTYRA316−37.42010.1488.6881.0077.38C
ATOM3332OHTYRA316−37.0159.0617.9791.0077.21O
ATOM3333NVALA317−39.05715.91012.8011.0074.20N
ATOM3334CAVALA317−39.87216.93413.4661.0072.51C
ATOM3335CVALA317−39.31618.35513.3301.0072.64C
ATOM3336OVALA317−40.08619.31113.2531.0072.17O
ATOM3337CBVALA317−40.08816.56014.9841.0072.14C
ATOM3338CG1VALA317−40.33617.76015.8041.0074.78C
ATOM3339CG2VALA317−41.25315.60315.1111.0069.59C
ATOM3340NASNA318−37.98818.49113.2961.0074.61N
ATOM3341CAASNA318−37.34219.79313.0781.0075.44C
ATOM3342CASNA318−37.88220.54811.8571.0075.25C
ATOM3343OASNA318−37.91221.77911.8441.0072.63O
ATOM3344CBASNA318−35.84319.64512.9101.0074.36C
ATOM3345CGASNA318−35.17120.95812.6351.0079.05C
ATOM3346OD1ASNA318−35.02321.80613.5221.0080.80O
ATOM3347ND2ASNA318−34.76121.14211.3961.0083.57N
ATOM3348NSERA319−38.30819.79810.8361.0075.32N
ATOM3349CASERA319−38.95120.3659.6401.0076.04C
ATOM3350CSERA319−40.21821.1949.9251.0076.24C
ATOM3351OSERA319−40.67021.9309.0461.0077.32O
ATOM3352CBSERA319−39.28519.2458.6351.0074.82C
ATOM3353OGSERA319−38.12018.5098.2811.0077.99O
ATOM3354NGLYA320−40.78221.07811.1331.0075.25N
ATOM3355CAGLYA320−41.93421.85711.5441.0074.43C
ATOM3356CGLYA320−41.64223.11012.3501.0076.72C
ATOM3357OGLYA320−42.56223.88412.5811.0078.81O
ATOM3358NPHEA321−40.39123.32812.7771.0075.79N
ATOM3359CAPHEA321−40.07624.44013.7061.0077.01C
ATOM3360CPHEA321−39.76125.78013.1121.0077.35C
ATOM3361OPHEA321−40.17826.79913.6801.0075.78O
ATOM3362CBPHEA321−38.91224.05114.6341.0078.53C
ATOM3363CGPHEA321−39.19222.85115.4971.0078.83C
ATOM3364CD1PHEA321−38.15422.00515.8571.0082.01C
ATOM3365CD2PHEA321−40.49022.55115.9531.0081.40C
ATOM3366CE1PHEA321−38.38320.89116.6501.0081.76C
ATOM3367CE2PHEA321−40.72221.44616.7401.0083.47C
ATOM3368CZPHEA321−39.66720.61417.0911.0085.30C
ATOM3369NASNA322−39.04025.80711.9961.0077.86N
ATOM3370CAASNA322−38.63727.08211.3811.0078.02C
ATOM3371CASNA322−39.78528.06111.1421.0078.43C
ATOM3372OASNA322−39.62029.23711.4401.0077.20O
ATOM3373CBASNA322−37.86626.85610.0871.0079.19C
ATOM3374CGASNA322−36.53126.20810.3151.0081.79C
ATOM3375OD1ASNA322−36.15825.90111.4551.0074.61O
ATOM3376ND2ASNA322−35.79125.9899.2291.0085.04N
ATOM3377NPROA323−40.94127.58210.6091.0078.55N
ATOM3378CAPROA323−42.08828.47910.4691.0079.23C
ATOM3379CPROA323−42.46929.17911.7791.0080.55C
ATOM3380OPROA323−42.79530.36711.7511.0083.61O
ATOM3381CBPROA323−43.20027.5429.9971.0080.04C
ATOM3382CGPROA323−42.47426.4739.2661.0079.60C
ATOM3383CDPROA323−41.27126.23210.1041.0078.83C
ATOM3384NLEUA324−42.42628.45412.8991.0080.72N
ATOM3385CALEUA324−42.68929.05014.2331.0081.22C
ATOM3386CLEUA324−41.58030.01314.6511.0078.60C
ATOM3387OLEUA324−41.85131.05315.2301.0077.91O
ATOM3388CBLEUA324−42.84727.97015.3101.0083.48C
ATOM3389CGLEUA324−44.01727.00315.1251.0090.04C
ATOM3390CD1LEUA324−43.82125.72715.9481.0091.90C
ATOM3391CD2LEUA324−45.32927.70715.4741.0093.47C
ATOM3392NILEA325−40.33429.65614.3551.0077.22N
ATOM3393CAILEA325−39.18630.50414.6891.0076.61C
ATOM3394CILEA325−39.24431.83013.9071.0075.96C
ATOM3395OILEA325−38.79132.85014.4071.0076.16O
ATOM3396CBILEA325−37.83729.77614.4331.0076.87C
ATOM3397CG1ILEA325−37.70128.52015.3281.0077.05C
ATOM3398CG2ILEA325−36.65430.70114.6891.0073.66C
ATOM3399CD1ILEA325−36.61127.53614.8701.0070.46C
ATOM3400NTYRA326−39.80331.81512.6901.0076.28N
ATOM3401CATYRA326−39.93433.05411.8841.0076.44C
ATOM3402CTYRA326−40.97834.01712.4161.0073.86C
ATOM3403OTYRA326−40.99135.15311.9921.0077.86O
ATOM3404CBTYRA326−40.24232.77210.4091.0077.72C
ATOM3405CGTYRA326−39.24031.8939.7011.0077.26C
ATOM3406CD1TYRA326−39.66630.8898.8471.0077.44C
ATOM3407CD2TYRA326−37.86532.0579.8851.0081.33C
ATOM3408CE1TYRA326−38.76430.0778.1941.0078.66C
ATOM3409CE2TYRA326−36.96131.2489.2361.0083.58C
ATOM3410CZTYRA326−37.42330.2588.3891.0080.70C
ATOM3411OHTYRA326−36.54129.4517.7421.0081.84O
ATOM3412NCYSA327−41.84833.58213.3321.0072.88N
ATOM3413CACYSA327−42.78234.49613.9991.0076.25C
ATOM3414CCYSA327−42.06435.53714.9051.0078.27C
ATOM3415OCYSA327−42.68436.50815.3301.0079.91O
ATOM3416CBCYSA327−43.82733.72914.8051.0077.89C
ATOM3417SGCYSA327−44.97032.74213.7781.0082.26S
ATOM3418NARGA328−40.77035.32015.1911.0079.14N
ATOM3419CAARGA328−39.91936.32415.8321.0079.59C
ATOM3420CARGA328−39.78137.59614.9841.0079.99C
ATOM3421OARGA328−39.58138.68015.5241.0079.93O
ATOM3422CBARGA328−38.51535.77116.0781.0077.25C
ATOM3423CGARGA328−38.42334.64617.1301.0076.09C
ATOM3424CDARGA328−36.99834.06717.2201.0077.31C
ATOM3425NEARGA328−36.00635.12917.0481.0075.58N
ATOM3426CZARGA328−35.53735.94517.9851.0081.23C
ATOM3427NH1ARGA328−35.95335.85919.2521.0074.46N
ATOM3428NH2ARGA328−34.63136.86617.6401.0081.80N
ATOM3429NSERA329−39.88537.44913.6591.0082.57N
ATOM3430CASERA329−39.78838.57412.7431.0084.20C
ATOM3431CSERA329−41.10739.32412.7111.0084.79C
ATOM3432OSERA329−42.15138.68712.5971.0088.37O
ATOM3433CBSERA329−39.46038.09711.3351.0084.66C
ATOM3434OGSERA329−39.46539.19010.4291.0090.44O
ATOM3435NPROA330−41.07640.67512.8141.0086.62N
ATOM3436CAPROA330−42.33941.41512.6851.0085.84C
ATOM3437CPROA330−42.90541.36911.2531.0086.45C
ATOM3438OPROA330−44.10341.46611.0791.0085.67O
ATOM3439CBPROA330−41.96042.85113.0801.0085.22C
ATOM3440CGPROA330−40.59742.76413.6691.0085.83C
ATOM3441CDPROA330−39.94641.59513.0451.0085.56C
ATOM3442NASPA331−42.03141.21610.2561.0087.75N
ATOM3443CAASPA331−42.44041.1278.8541.0088.35C
ATOM3444CASPA331−43.16039.8318.5161.0088.54C
ATOM3445OASPA331−44.18039.8657.8241.0087.13O
ATOM3446CBASPA331−41.22541.2687.9291.0091.75C
ATOM3447CGASPA331−40.58542.6408.0001.0099.65C
ATOM3448OD1ASPA331−41.15243.5268.6601.00101.57O
ATOM3449OD2ASPA331−39.50742.8257.3871.00108.72O
ATOM3450NPHEA332−42.63838.6978.9921.0087.91N
ATOM3451CAPHEA332−43.30637.3878.7591.0085.83C
ATOM3452CPHEA332−44.59637.2989.5251.0084.97C
ATOM3453OPHEA332−45.58336.8119.0061.0085.85O
ATOM3454CBPHEA332−42.41936.2069.1421.0083.02C
ATOM3455CGPHEA332−41.42135.8488.0991.0081.58C
ATOM3456CD1PHEA332−40.18436.4708.0641.0079.60C
ATOM3457CD2PHEA332−41.71934.8847.1411.0082.96C
ATOM3458CE1PHEA332−39.25136.1387.0921.0083.90C
ATOM3459CE2PHEA332−40.79434.5416.1591.0081.77C
ATOM3460CZPHEA332−39.55835.1666.1321.0084.78C
ATOM3461NARGA333−44.56737.77210.7641.0085.64N
ATOM3462CAARGA333−45.74237.79511.6181.0086.23C
ATOM3463CARGA333−46.87538.60310.9641.0085.68C
ATOM3464OARGA333−48.01538.17111.0051.0085.47O
ATOM3465CBARGA333−45.35738.36212.9631.0086.76C
ATOM3466CGARGA333−46.38338.22214.0591.0090.70C
ATOM3467CDARGA333−45.67138.26315.4071.0093.90C
ATOM3468NEARGA333−44.71039.37715.4721.00100.54N
ATOM3469CZARGA333−43.69339.49016.3351.00103.24C
ATOM3470NH1ARGA333−43.44938.55317.2621.00104.89N
ATOM3471NH2ARGA333−42.89940.56616.2711.00102.64N
ATOM3472NILEA334−46.53439.75510.3731.0084.51N
ATOM3473CAILEA334−47.47240.5809.5841.0083.97C
ATOM3474CILEA334−47.89839.8628.3161.0082.02C
ATOM3475OILEA334−49.07739.8717.9631.0086.53O
ATOM3476CBILEA334−46.86241.9599.2111.0085.26C
ATOM3477CG1ILEA334−46.77742.86010.4441.0085.90C
ATOM3478CG2ILEA334−47.69242.6688.1421.0082.88C
ATOM3479CD1ILEA334−45.79144.01510.2871.0088.19C
ATOM3480NALAA335−46.93439.2487.6351.0080.09N
ATOM3481CAALAA335−47.20238.4706.4331.0079.44C
ATOM3482CALAA335−48.19937.3456.7191.0078.78C
ATOM3483OALAA335−49.20737.2516.0571.0078.15O
ATOM3484CBALAA335−45.91137.8965.8761.0078.92C
ATOM3485NPHEA336−47.89636.5117.7141.0078.39N
ATOM3486CAPHEA336−48.76035.3728.0921.0077.87C
ATOM3487CPHEA336−50.18435.7518.4271.0077.70C
ATOM3488OPHEA336−51.11635.2057.8471.0075.76O
ATOM3489CBPHEA336−48.19834.6029.3011.0077.26C
ATOM3490CGPHEA336−46.85233.9439.0721.0077.36C
ATOM3491CD1PHEA336−46.40833.5627.7961.0078.61C
ATOM3492CD2PHEA336−46.02433.69510.1521.0076.74C
ATOM3493CE1PHEA336−45.18032.9627.6211.0076.21C
ATOM3494CE2PHEA336−44.78733.0899.9741.0078.36C
ATOM3495CZPHEA336−44.37232.7248.7001.0076.78C
ATOM3496NGLNA337−50.34636.6819.3631.0078.23N
ATOM3497CAGLNA337−51.69137.1249.7921.0079.51C
ATOM3498CGLNA337−52.54937.7118.6421.0078.03C
ATOM3499OGLNA337−53.77837.6468.6941.0078.24O
ATOM3500CBGLNA337−51.60938.08110.9971.0080.74C
ATOM3501CGGLNA337−50.87039.39110.8021.0086.86C
ATOM3502CDGLNA337−50.61340.11312.1431.0089.20C
ATOM3503OE1GLNA337−51.31739.87713.1271.0097.34O
ATOM3504NE2GLNA337−49.61040.98912.1771.0094.24N
ATOM3505NGLUA338−51.90038.2717.6221.0076.38N
ATOM3506CAGLUA338−52.59138.6946.4111.0074.47C
ATOM3507CGLUA338−53.08537.4485.6701.0073.86C
ATOM3508OGLUA338−54.25837.3705.2821.0073.06O
ATOM3509CBGLUA338−51.65939.5125.5161.0075.29C
ATOM3510CGGLUA338−52.34340.2794.3651.0079.56C
ATOM3511CDGLUA338−52.69539.4423.1131.0085.28C
ATOM3512OE1GLUA338−52.50038.2073.0871.0091.20O
ATOM3513OE2GLUA338−53.17640.0482.1311.0089.12O
ATOM3514NLEUA339−52.17736.4885.4771.0070.47N
ATOM3515CALEUA339−52.47535.2424.7631.0071.14C
ATOM3516CLEUA339−53.52734.3755.4631.0072.95C
ATOM3517OLEUA339−54.25533.6504.7931.0074.46O
ATOM3518CBLEUA339−51.20934.4064.5671.0068.65C
ATOM3519CGLEUA339−50.05535.0213.7781.0069.52C
ATOM3520CD1LEUA339−48.80534.1393.9351.0068.79C
ATOM3521CD2LEUA339−50.41135.2252.3151.0062.62C
ATOM3522NLEUA340−53.59934.4556.7961.0074.28N
ATOM3523CALEUA340−54.59833.7187.5881.0075.08C
ATOM3524CLEUA340−55.84834.5737.9011.0076.80C
ATOM3525OLEUA340−56.57334.2808.8601.0076.39O
ATOM3526CBLEUA340−53.96033.1748.8781.0074.68C
ATOM3527CGLEUA340−52.76332.2338.7011.0073.84C
ATOM3528CD1LEUA340−52.15031.88710.0481.0073.83C
ATOM3529CD2LEUA340−53.16730.9767.9821.0073.24C
ATOM3530NCYSA341−56.08335.6197.0881.0079.59N
ATOM3531CACYSA341−57.28036.4777.1521.0080.75C
ATOM3532CCYSA341−57.53637.0898.5301.0081.21C
ATOM3533OCYSA341−58.61336.9119.1141.0081.97O
ATOM3534CBCYSA341−58.50035.6836.6741.0081.26C
ATOM3535SGCYSA341−58.24334.8895.0791.0088.24S
ATOM3536NLEUA342−56.53337.8099.0311.0081.84N
ATOM3537CALEUA342−56.59838.48310.3331.0082.60C
ATOM3538CLEUA342−56.37039.99310.1581.0083.35C
ATOM3539OLEUA342−55.71540.65010.9711.0085.10O
ATOM3540CBLEUA342−55.57537.87811.3031.0082.19C
ATOM3541CGLEUA342−55.51336.34711.4071.0082.55C
ATOM3542CD1LEUA342−54.41535.91312.3681.0082.95C
ATOM3543CD2LEUA342−56.85135.78211.8381.0083.45C
TER3544LEUA342
HETATM3545C1MALA401−30.43367.55122.6041.00116.54C
HETATM3546C2MALA401−29.50868.27121.6131.00116.67C
HETATM3547C3MALA401−28.28667.46621.2501.00115.67C
HETATM3548C4MALA401−27.43767.77422.4751.00113.35C
HETATM3549C5MALA401−28.22267.32623.7421.00114.16C
HETATM3550C6MALA401−27.46767.82124.9971.00112.25C
HETATM3551O1MALA401−30.86066.15222.4811.00118.82O
HETATM3552O2MALA401−30.19668.89620.4301.00113.17O
HETATM3553O3MALA401−27.69267.92320.0421.00118.88O
HETATM3554O4MALA401−26.15167.13322.4171.00110.96O
HETATM3555O5MALA401−29.64067.72323.7961.00116.03O
HETATM3556O6MALA401−28.32968.19126.0911.00111.18O
HETATM3557C1′MALA401−34.22564.36524.5551.00118.64C
HETATM3558C2′MALA401−34.06265.87524.6951.00118.98C
HETATM3559C3′MALA401−33.05366.49723.7321.00118.89C
HETATM3560C4′MALA401−31.77365.65323.5331.00118.41C
HETATM3561C5′MALA401−32.19264.16923.3291.00117.27C
HETATM3562C6′MALA401−31.04463.16423.1541.00114.49C
HETATM3563O1′MALA401−34.90563.85025.7051.00115.98O
HETATM3564O2′MALA401−35.32866.51024.4631.00120.41O
HETATM3565O3′MALA401−32.80367.77124.3461.00118.98O
HETATM3566O5′MALA401−32.95163.73724.4671.00118.26O
HETATM3567O6′MALA401−31.31961.88923.7811.00101.09O
HETATM3568SSO4A402−30.54940.5871.8401.0088.35S
HETATM3569O1SO4A402−31.01240.6570.4491.0091.49O
HETATM3570O2SO4A402−31.34141.5182.6481.0079.98O
HETATM3571O3SO4A402−30.74739.2332.3891.0086.89O
HETATM3572O4SO4A402−29.13640.9421.8081.0079.16O
HETATM3573SSO4A403−34.52939.15420.8411.0091.42S
HETATM3574O1SO4A403−35.51840.19321.1311.0084.45O
HETATM3575O2SO4A403−33.29639.33421.6121.0093.22O
HETATM3576O3SO4A403−35.07837.87521.2321.0084.98O
HETATM3577O4SO4A403−34.19339.24619.4381.0095.59O
HETATM3578SSO4A404−26.67761.41043.6671.0079.61S
HETATM3579O1SO4A404−26.22460.48242.6371.0081.00O
HETATM3580O2SO4A404−27.96862.00143.3151.0078.07O
HETATM3581O3SO4A404−26.85260.65544.9071.0085.67O
HETATM3582O4SO4A404−25.70062.48743.8191.0076.33O
HETATM3583SSO4A405−38.22254.16728.6131.0076.15S
HETATM3584O1SO4A405−36.81554.45428.3711.0062.35O
HETATM3585O2SO4A405−38.70853.20227.6281.0074.38O
HETATM3586O3SO4A405−38.39253.56129.9251.0077.47O
HETATM3587O4SO4A405−38.98355.41228.5361.0074.20O
HETATM3588SSO4A406−14.62646.74234.9551.00133.97S
HETATM3589O1SO4A406−14.90546.88833.5271.00132.94O
HETATM3590O2SO4A406−15.87046.48535.6901.00131.52O
HETATM3591O3SO4A406−13.74445.58835.1411.00137.48O
HETATM3592O4SO4A406−13.96847.95335.4601.00129.69O
HETATM3593SSO4A407−39.37559.24210.9571.00103.03S
HETATM3594O1SO4A407−38.11258.59110.5891.00100.34O
HETATM3595O2SO4A407−40.36559.0299.8991.00104.55O
HETATM3596O3SO4A407−39.86558.66312.2111.00107.47O
HETATM3597O4SO4A407−39.17460.68311.1381.00106.88O
HETATM3598O17CAUA408−33.47710.9578.1701.0050.96O
HETATM3599C16CAUA408−32.26710.2308.0411.0045.65C
HETATM3600C18CAUA408−32.4788.9517.2251.0051.24C
HETATM3601N19CAUA408−33.7028.2507.6001.0054.99N
HETATM3602C20CAUA408−33.8066.8057.4981.0060.13C
HETATM3603C21CAUA408−33.5336.3856.0551.0066.62C
HETATM3604C22CAUA408−35.1846.3507.9881.0059.87C
HETATM3605C15CAUA408−31.24211.1057.3641.0046.24C
HETATM3606O14CAUA408−30.04910.3677.1821.0051.01O
HETATM3607C13CAUA408−28.93110.8576.5811.0052.29C
HETATM3608C12CAUA408−28.91112.1336.0051.0056.44C
HETATM3609C11CAUA408−27.76812.6285.3931.0056.29C
HETATM3610C10CAUA408−26.60811.8755.3321.0054.90C
HETATM3611C8CAUA408−26.56510.6255.8751.0054.68C
HETATM3612C9CAUA408−27.76810.1086.5171.0053.44C
HETATM3613N7CAUA408−25.5939.6575.9741.0057.07N
HETATM3614C3CAUA408−26.0968.5616.6371.0055.09C
HETATM3615C4CAUA408−27.4828.8636.9761.0053.66C
HETATM3616C2CAUA408−25.5897.3387.0091.0054.54C
HETATM3617C1CAUA408−26.3956.4327.6891.0056.87C
HETATM3618C6CAUA408−27.7176.7318.0061.0056.28C
HETATM3619C5CAUA408−28.2697.9487.6521.0056.42C
HETATM3620C1BU1A409−26.14241.42419.1271.0052.74C
HETATM3621C2BU1A409−24.67341.19419.3491.0050.50C
HETATM3622C3BU1A409−24.36139.70319.2561.0058.68C
HETATM3623C4BU1A409−23.63939.19320.4921.0060.90C
HETATM3624O5BU1A409−26.44942.81219.0631.0046.89O
HETATM3625O6BU1A409−23.39737.79620.3581.0065.39O
HETATM3626C1BU1A410−25.49226.52317.7271.0080.96C
HETATM3627C2BU1A410−25.25926.81816.2401.0075.39C
HETATM3628C3BU1A410−25.56928.28615.9041.0072.90C
HETATM3629C4BU1A410−24.36829.07615.3931.0070.20C
HETATM3630O5BU1A410−25.03625.20218.0961.0082.37O
HETATM3631O6BU1A410−23.72829.76916.4681.0072.23O
HETATM3632C1ACMA411−33.36545.11623.7751.0072.09C
HETATM3633OACMA411−34.58045.10023.7841.0074.74O
HETATM3634NACMA411−32.67743.98123.8971.0071.73N
HETATM3635C2ACMA411−32.58746.39723.6261.0069.99C
HETATM3636C1CLRA412−36.29227.653−4.3521.0098.97C
HETATM3637C2CLRA412−36.57129.133−4.5911.0098.10C
HETATM3638C3CLRA412−35.42529.793−5.3541.0098.91C
HETATM3639C4CLRA412−34.96529.038−6.6271.0098.80C
HETATM3640C5CLRA412−35.00127.515−6.5131.0099.38C
HETATM3641C6CLRA412−34.08826.785−7.2091.0099.72C
HETATM3642C7CLRA412−34.01725.259−7.2041.0098.91C
HETATM3643C8CLRA412−35.27524.631−6.6331.0099.40C
HETATM3644C9CLRA412−35.65125.377−5.3371.0099.32C
HETATM3645C10CLRA412−36.05026.846−5.6451.00100.31C
HETATM3646C11CLRA412−36.70724.655−4.4661.0096.50C
HETATM3647C12CLRA412−36.53623.143−4.3651.0096.65C
HETATM3648C13CLRA412−36.32922.495−5.7191.00100.10C
HETATM3649C14CLRA412−35.10023.138−6.3531.00101.09C
HETATM3650C15CLRA412−34.78222.237−7.5421.00101.42C
HETATM3651C16CLRA412−35.21720.837−7.0811.00101.37C
HETATM3652C17CLRA412−35.99521.000−5.7581.00100.60C
HETATM3653C18CLRA412−37.56222.726−6.5711.00103.09C
HETATM3654C19CLRA412−37.26826.936−6.5121.00102.37C
HETATM3655C20CLRA412−37.14419.961−5.6321.00100.25C
HETATM3656C21CLRA412−38.03220.187−4.4061.0098.50C
HETATM3657C22CLRA412−36.56118.538−5.6571.00101.01C
HETATM3658C23CLRA412−37.43717.457−5.0121.00103.22C
HETATM3659C24CLRA412−36.74816.094−5.0991.00105.36C
HETATM3660C25CLRA412−37.53515.014−5.8531.00107.48C
HETATM3661C26CLRA412−36.57114.080−6.5911.00107.73C
HETATM3662C27CLRA412−38.40214.192−4.8991.00106.61C
HETATM3663O1CLRA412−35.82831.158−5.6641.0099.70O
HETATM3664C1CLRA413−52.08329.4423.1981.0084.76C
HETATM3665C2CLRA413−52.24430.9513.1001.0085.75C
HETATM3666C3CLRA413−53.21431.2961.9561.0084.48C
HETATM3667C4CLRA413−54.60630.7182.2331.0082.59C
HETATM3668C5CLRA413−54.42429.2182.2451.0086.97C
HETATM3669C6CLRA413−55.17128.4681.4001.0086.01C
HETATM3670C7CLRA413−55.07726.9681.3091.0088.00C
HETATM3671C8CLRA413−54.34026.3622.4871.0089.33C
HETATM3672C9CLRA413−53.08727.1612.8621.0088.72C
HETATM3673C10CLRA413−53.39928.6363.2141.0085.54C
HETATM3674C11CLRA413−52.26526.4403.9591.0087.79C
HETATM3675C12CLRA413−51.96124.9813.6161.0090.38C
HETATM3676C13CLRA413−53.21924.2023.2831.0090.23C
HETATM3677C14CLRA413−53.90424.9332.1421.0092.32C
HETATM3678C15CLRA413−54.97623.9711.6681.0092.65C
HETATM3679C16CLRA413−54.28222.6161.8271.0093.79C
HETATM3680C17CLRA413−53.01822.8202.6931.0091.53C
HETATM3681C18CLRA413−54.15624.0934.4991.0087.86C
HETATM3682C19CLRA413−54.03928.7744.5951.0082.64C
HETATM3683C20CLRA413−52.77221.6243.6531.0091.51C
HETATM3684C21CLRA413−51.81521.8784.8161.0089.13C
HETATM3685C22CLRA413−52.23320.4302.8571.0091.91C
HETATM3686C23CLRA413−52.84119.0993.2961.0093.30C
HETATM3687C24CLRA413−52.45917.9932.3111.0092.99C
HETATM3688C25CLRA413−51.93816.7463.0021.0092.39C
HETATM3689C26CLRA413−52.05915.5412.0761.0094.36C
HETATM3690C27CLRA413−50.49916.9523.4601.0089.59C
HETATM3691O1CLRA413−53.33432.6841.6071.0081.53O
HETATM3692C1CLRA414−41.70627.953−4.4191.00114.97C
HETATM3693C2CLRA414−41.76829.448−4.7091.00114.73C
HETATM3694C3CLRA414−40.66429.838−5.6941.00114.23C
HETATM3695C4CLRA414−40.71129.022−7.0001.00115.07C
HETATM3696C5CLRA414−40.83627.528−6.7141.00115.29C
HETATM3697C6CLRA414−40.06926.669−7.4181.00115.44C
HETATM3698C7CLRA414−40.08125.169−7.2261.00116.17C
HETATM3699C8CLRA414−41.30324.678−6.4731.00115.82C
HETATM3700C9CLRA414−41.56725.570−5.2521.00114.14C
HETATM3701C10CLRA414−41.81527.052−5.6521.00114.08C
HETATM3702C11CLRA414−42.68524.996−4.3481.00113.90C
HETATM3703C12CLRA414−42.45923.526−3.9991.00115.68C
HETATM3704C13CLRA414−42.29422.667−5.2381.00115.89C
HETATM3705C14CLRA414−41.12323.217−6.0421.00116.05C
HETATM3706C15CLRA414−40.93522.171−7.1431.00117.57C
HETATM3707C16CLRA414−41.28520.849−6.4501.00117.86C
HETATM3708C17CLRA414−41.87421.195−5.0601.00116.61C
HETATM3709C18CLRA414−43.60222.724−6.0511.00114.74C
HETATM3710C19CLRA414−43.19727.247−6.2851.00114.74C
HETATM3711C20CLRA414−42.93720.193−4.5161.00116.12C
HETATM3712C21CLRA414−43.28420.508−3.0651.00114.86C
HETATM3713C22CLRA414−42.50318.733−4.6031.00115.06C
HETATM3714C23CLRA414−43.58617.708−4.2161.00115.00C
HETATM3715C24CLRA414−42.98516.411−3.6621.00113.44C
HETATM3716C25CLRA414−42.27215.571−4.7261.00113.32C
HETATM3717C26CLRA414−40.86916.077−5.0181.00111.52C
HETATM3718C27CLRA414−42.20414.107−4.3091.00112.05C
HETATM3719O1CLRA414−40.67431.263−5.9661.00114.05O
HETATM3720C1PLMA415−57.95933.3034.9491.0094.70C
HETATM3721O2PLMA415−57.04733.7324.2111.0099.49O
HETATM3722C2PLMA415−58.01231.8285.2791.0093.74C
HETATM3723C3PLMA415−59.13131.1254.4921.0092.47C
HETATM3724C4PLMA415−58.64229.9823.5901.0091.37C
HETATM3725C5PLMA415−59.53228.7423.7011.0089.98C
HETATM3726C6PLMA415−59.31027.8162.5051.0088.93C
HETATM3727C7PLMA415−60.00326.4672.6771.0086.74C
HETATM3728C8PLMA415−59.01225.3302.4671.0086.47C
HETATM3729C9PLMA415−59.70923.9712.5041.0088.96C
HETATM3730CAPLMA415−58.83722.8933.1561.0090.43C
HETATM3731CBPLMA415−59.09421.4742.6221.0094.25C
HETATM3732CCPLMA415−57.86320.7752.0161.0096.09C
HETATM3733CDPLMA415−57.47119.4832.7521.00100.00C
HETATM3734CEPLMA415−56.63518.5441.8721.00101.35C
HETATM3735CFPLMA415−56.39117.2072.5761.00101.14C
HETATM3736CGPLMA415−55.69516.1991.6641.0099.15C
HETATM3737C3512PA416−12.91554.89717.8071.00109.60C
HETATM3738O3412PA416−13.94954.68318.7771.00110.80O
HETATM3739C3312PA416−13.45154.39420.0911.00108.13C
HETATM3740C3212PA416−13.56855.62220.9821.00105.37C
HETATM3741O3112PA416−12.29956.02221.5391.00101.39O
HETATM3742C3012PA416−12.28357.34022.1331.00102.00C
HETATM3743C2912PA416−13.23157.47623.3461.0099.93C
HETATM3744O2812PA416−14.58057.57522.8741.0094.22O
HETATM3745C2712PA416−15.58058.13223.7201.0091.37C
HETATM3746C2612PA416−16.52858.91222.8201.0089.49C
HETATM3747O2512PA416−15.75259.82522.0161.0091.31O
HETATM3748C2412PA416−16.08159.83320.6211.0089.90C
HETATM3749C2312PA416−14.99860.57419.8381.0088.02C
HETATM3750O2212PA416−14.83861.90420.3521.0086.45O
HETATM3751C2112PA416−15.83962.83819.9441.0077.75C
HETATM3752C2012PA416−15.14663.91619.1491.0082.56C
HETATM3753O1912PA416−14.00564.46219.8341.0079.81O
HETATM3754C1812PA416−14.32365.26320.9881.0086.00C
HETATM3755C1712PA416−13.22366.27521.3321.0087.84C
HETATM3756O1612PA416−11.99865.96720.6531.0093.31O
HETATM3757C1512PA416−11.01165.35421.4931.0096.79C
HETATM3758OHOHA501−23.20138.1681.1271.0060.95O
HETATM3759OHOHA502−28.32245.44519.1061.0062.27O
HETATM3760OHOHA503−25.66351.54011.5571.0073.63O
HETATM3761OHOHA504−18.40354.49020.5681.0049.22O
HETATM3762OHOHA505−28.86562.96714.4761.0049.07O
HETATM3763OHOHA506−38.34425.3956.9991.0053.60O
HETATM3764OHOHA507−24.23547.66928.2791.0058.12O
HETATM3765OHOHA508−29.56658.64429.5051.0053.32O
HETATM3766OHOHA509−9.12965.07735.7881.0053.82O
HETATM3767OHOHA510−31.58856.2355.2701.0054.83O
HETATM3768OHOHA511−33.767−0.9362.9701.0076.89O
HETATM3769OHOHA512−36.83152.16325.6861.0059.37O
HETATM3770OHOHA513−20.29351.7845.3691.0057.17O
HETATM3771OHOHA514−33.71048.03830.0531.0055.19O
HETATM3772OHOHA515−27.49871.73731.0971.0072.77O
HETATM3773OHOHA516−38.41975.00317.4091.0063.18O
HETATM3774OHOHA517−13.00852.29133.7201.0054.29O
HETATM3775OHOHA518−25.41749.4492.5261.0084.88O
HETATM3776OHOHA519−44.49224.10310.5941.0070.04O
HETATM3777OHOHA520−53.19234.656−0.0120.5068.32O
HETATM3778OHOHA521−33.73052.33413.5311.0071.71O
HETATM3779OHOHA522−19.40144.89411.2841.0082.98O
HETATM3780OHOHA523−20.706−4.4555.0291.0074.00O
HETATM3781OHOHA524−18.87844.84324.4351.0071.79O
HETATM3782OHOHA525−26.26647.54612.0741.0096.88O
HETATM3783OHOHA526−35.81854.61225.4481.0065.23O
HETATM3784OHOHA527−27.91163.92817.0981.0056.73O
HETATM3785OHOHA528−34.13624.30012.3741.0064.85O
HETATM3786OHOHA529−31.48513.40316.4271.0055.45O
HETATM3787OHOHA530−33.22946.52527.5091.0062.72O
HETATM3788OHOHA531−37.00056.92726.4701.0069.85O
HETATM3789OHOHA532−38.27223.6919.9911.0074.37O
HETATM3790OHOHA533−23.73845.70030.2141.0070.87O
HETATM3791OHOHA534−35.54718.9179.3681.0068.76O
HETATM3792OHOHA535−27.52046.01835.7091.0073.73O
HETATM3793OHOHA536−11.16952.63927.1071.0053.63O
HETATM3794OHOHA537−35.16136.6036.9201.0084.70O
HETATM3795OHOHA538−13.33164.63116.8261.0078.41O
HETATM3796OHOHA539−15.73737.98913.5011.0074.20O
HETATM3797OHOHA540−17.61249.88519.7411.0067.69O
HETATM3798OHOHA541−28.87156.0414.9301.0062.51O
HETATM3799OHOHA542−18.10062.47017.1921.0064.11O
HETATM3800OHOHA543−39.8765.50210.3381.0078.73O
HETATM3801OHOHA544−39.732−5.317−4.5311.0075.67O
HETATM3802OHOHA545−19.86559.62117.2221.0067.78O
HETATM3803OHOHA546−37.39727.6726.1201.0059.62O
HETATM3804OHOHA547−19.19949.83344.4101.0055.49O
HETATM3805OHOHA548−35.61821.6458.8271.0061.27O
CONECT5971283
CONECT12291277
CONECT12771229
CONECT1283597
CONECT29063635
CONECT35353720
CONECT3545354635513555
CONECT3546354535473552
CONECT3547354635483553
CONECT3548354735493554
CONECT3549354835503555
CONECT355035493556
CONECT355135453560
CONECT35523546
CONECT35533547
CONECT35543548
CONECT355535453549
CONECT35563550
CONECT3557355835633566
CONECT3558355735593564
CONECT3559355835603565
CONECT3560355135593561
CONECT3561356035623566
CONECT356235613567
CONECT35633557
CONECT35643558
CONECT35653559
CONECT356635573561
CONECT35673562
CONECT35683569357035713572
CONECT35693568
CONECT35703568
CONECT35713568
CONECT35723568
CONECT35733574357535763577
CONECT35743573
CONECT35753573
CONECT35763573
CONECT35773573
CONECT35783579358035813582
CONECT35793578
CONECT35803578
CONECT35813578
CONECT35823578
CONECT35833584358535863587
CONECT35843583
CONECT35853583
CONECT35863583
CONECT35873583
CONECT35883589359035913592
CONECT35893588
CONECT35903588
CONECT35913588
CONECT35923588
CONECT35933594359535963597
CONECT35943593
CONECT35953593
CONECT35963593
CONECT35973593
CONECT35983599
CONECT3599359836003605
CONECT360035993601
CONECT360136003602
CONECT3602360136033604
CONECT36033602
CONECT36043602
CONECT360535993606
CONECT360636053607
CONECT3607360636083612
CONECT360836073609
CONECT360936083610
CONECT361036093611
CONECT3611361036123613
CONECT3612360736113615
CONECT361336113614
CONECT3614361336153616
CONECT3615361236143619
CONECT361636143617
CONECT361736163618
CONECT361836173619
CONECT361936153618
CONECT362036213624
CONECT362136203622
CONECT362236213623
CONECT362336223625
CONECT36243620
CONECT36253623
CONECT362636273630
CONECT362736263628
CONECT362836273629
CONECT362936283631
CONECT36303626
CONECT36313629
CONECT3632363336343635
CONECT36333632
CONECT36343632
CONECT363529063632
CONECT363636373645
CONECT363736363638
CONECT3638363736393663
CONECT363936383640
CONECT3640363936413645
CONECT364136403642
CONECT364236413643
CONECT3643364236443649
CONECT3644364336453646
CONECT36453636364036443654
CONECT364636443647
CONECT364736463648
CONECT36483647364936523653
CONECT3649364336483650
CONECT365036493651
CONECT365136503652
CONECT3652364836513655
CONECT36533648
CONECT36543645
CONECT3655365236563657
CONECT36563655
CONECT365736553658
CONECT365836573659
CONECT365936583660
CONECT3660365936613662
CONECT36613660
CONECT36623660
CONECT36633638
CONECT366436653673
CONECT366536643666
CONECT3666366536673691
CONECT366736663668
CONECT3668366736693673
CONECT366936683670
CONECT367036693671
CONECT3671367036723677
CONECT3672367136733674
CONECT36733664366836723682
CONECT367436723675
CONECT367536743676
CONECT36763675367736803681
CONECT3677367136763678
CONECT367836773679
CONECT367936783680
CONECT3680367636793683
CONECT36813676
CONECT36823673
CONECT3683368036843685
CONECT36843683
CONECT368536833686
CONECT368636853687
CONECT368736863688
CONECT3688368736893690
CONECT36893688
CONECT36903688
CONECT36913666
CONECT369236933701
CONECT369336923694
CONECT3694369336953719
CONECT369536943696
CONECT3696369536973701
CONECT369736963698
CONECT369836973699
CONECT3699369837003705
CONECT3700369937013702
CONECT37013692369637003710
CONECT370237003703
CONECT370337023704
CONECT37043703370537083709
CONECT3705369937043706
CONECT370637053707
CONECT370737063708
CONECT3708370437073711
CONECT37093704
CONECT37103701
CONECT3711370837123713
CONECT37123711
CONECT371337113714
CONECT371437133715
CONECT371537143716
CONECT3716371537173718
CONECT37173716
CONECT37183716
CONECT37193694
CONECT3720353537213722
CONECT37213720
CONECT372237203723
CONECT372337223724
CONECT372437233725
CONECT372537243726
CONECT372637253727
CONECT372737263728
CONECT372837273729
CONECT372937283730
CONECT373037293731
CONECT373137303732
CONECT373237313733
CONECT373337323734
CONECT373437333735
CONECT373537343736
CONECT37363735
CONECT37373738
CONECT373837373739
CONECT373937383740
CONECT374037393741
CONECT374137403742
CONECT374237413743
CONECT374337423744
CONECT374437433745
CONECT374537443746
CONECT374637453747
CONECT374737463748
CONECT374837473749
CONECT374937483750
CONECT375037493751
CONECT375137503752
CONECT375237513753
CONECT375337523754
CONECT375437533755
CONECT375537543756
CONECT375637553757
CONECT37573756
MASTER45501619401863804121939
END