Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nature. Author manuscript; available in PMC 2013 July 3.
Published in final edited form as:
PMCID: PMC3700570

Structure of the Chemokine Receptor CXCR1 in Phospholipid Bilayers


CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumor growth1-3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signaling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class-A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors4-7. Despite its importance, its molecular mechanism is poorly understood due to the limited structural information available. Recently, structure determination of GPCRs has advanced by tailoring the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are critical for G-protein interactions11 and activation of CXCR1 involves both N-terminal residues and extracellular loops2,12,13. Our previous NMR studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed.

To examine the structure and function of CXCR1 in its natural environment, we reconstituted the full-length, active receptor in phospholipid bilayers (proteoliposomes). The NMR method we developed, rotationally aligned (RA) solid-state NMR15, is specifically tailored for the unique properties of membrane proteins in liquid crystalline phospholipid bilayers. It combines features of magic angle spinning (MAS)16 and oriented sample (OS)17 solid-state NMR to resolve and assign resonances associated with each amino acid residue, measure site-specific orientation restraints relative to the bilayer, and calculate the three-dimensional structure of the protein and its integral membrane orientation. It differs from previously used OS methods because it relies on the inherent rotational diffusion of membrane proteins in phospholipid bilayers18 to provide orientation-dependent motional averaging of dipolar coupling (DC) powder patterns relative to the bilayer normal rather than the orientation-dependent frequencies of single-line resonances observed in OS NMR of stationary, uniaxially aligned samples. Furthermore, the method takes advantage of recent bioinformatics developments that facilitate molecular fragment replacement approaches to structure determination, including membrane proteins19-21.

CXCR1 was uniformly 13C/15N labeled by expression in Escherichia coli, then purified, refolded in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) proteoliposomes22,23, and placed as a concentrated suspension in a MAS rotor. Refolded CXCR1 binds IL-8 with high affinity (dissociation constant, Kd ~ 1–5 nM) and couples to its G-protein, Gi/o (half maximum effective concentration, EC50 ~ 1 nM)22, indicating that the NMR sample conditions are compatible with physiological activity (Supplementary Fig. 1).

As expected, the two-dimensional 13C/13C correlation spectrum of this 350-residue protein is quite crowded (Supplementary Fig. 2). However, expansion of the 13CA resonance region has sufficient resolution to contribute to the assignment process (Supplementary Fig. 2d). Spectra, obtained from a uniformly 13C labeled sample with a shorter mixing time and from a sample labeled using 2-13C-glycerol, have fewer signals and improved resolution. Regardless, the vast majority of data used to resolve, assign and measure isotropic chemical shift frequencies from N, CA, CO, and CB sites were obtained from 13C-detected three-dimensional, triple-resonance experiments (Fig. 1b; Supplementary Figs. 3-4; Supplementary Table 2). Overall, 97% of the backbone resonances for residues 20 to 325 were assigned. The missing resonances are from seven Pro (P22, P93, P170, P180, P185, P214, P257) and one Arg (R285). None of the 15N and 13C signals from the mobile N-and C-termini (residues 1-19 and 326-350) could be detected in the spectra, consistent with our observation of these signals in solid-state NMR experiments designed to detect only signals from mobile sites (Supplementary Fig. 6), as well as our previous analysis of local and global motions of CXCR124.

Figure 1
Structure determination of CXCR1

Three-dimensional 13C-detected separated local field (SLF) experiments15 were used to measure the 1H-15N DC and 1H-13CA DC frequencies that provide orientation restraints for structure determination (Fig. 1c, 1d; Supplementary Fig. 5). The protein backbone structure was calculated by a molecular fragment replacement approach. An initial structural model was generated from a set of molecular fragments generated with CS-Rosetta20 from the experimental chemical shifts, the amino acid sequence of CXCR1, and the helical framework of the prototypical class A GPCR bovine rhodopsin25. This initial model was first refined with the experimental restraints using the all atom19 and the implicit membrane21 potentials of Rosetta. Finally, the resulting structural model was refined by restrained simulated annealing using Xplor-NIH26.

The three-dimensional structure of CXCR1 (Fig. 1e) has the consensus fold of a GPCR, with seven transmembrane helices (TM1-TM7) connected by three extracellular loops (ECL1-ECL3) and three intracellular loops (ICL1-ICL3). The average backbone pairwise RMSD is 1.7 Å (Supplementary Table 1) and the experimentally measured 1H-15N DC and 1H-13CA DC values correlate remarkably well with those calculated from the refined protein structure (Fig. 1g; Supplementary Fig. 7). Notably, the correlations improve dramatically following refinement of the initial structural model with the experimental data, demonstrating that the NMR structure of CXCR1 is determined by the experimentally measured backbone orientation restraints and dihedral angles. We anticipate that both structural accuracy and precision will improve with inclusion of side chain restraints and, where feasible, distance restraints.

DCs contain information about molecular orientation as well as dynamics, and scaling of their values by local internal motions would compromise their analysis in terms of pure orientation restraints. For CXCR1, analysis of the DC data yielded similar values of the magnitude and symmetry of the molecular order tensor for residues in the helices and loops, indicating that these regions of the protein experience a similar degree of order in the lipid bilayer. This is supported by the excellent fit of the DC correlation plots obtained with a single value of the order tensor (Fig. 1g; Supplementary Fig. 7b) and by the observation that >20% of the 1H-15N DC signals from residues distributed throughout the protein sequence, including in loop sites, fall within 10% of the maximum value (21 kHz) expected for a static crystalline sample (Fig. 1d).

Following the structure determination of rhodopsin from three- and two-dimensional crystals25,27, the structures of several class A ligand-activated GPCRs have recently been determined by X-ray crystallography6,7. CXCR1 is now the first GPCR with its structure determined in liquid crystalline phospholipid bilayers, and the first ligand-activated GPCR with its structure determined without modification of its amino acid sequence. The structure of CXCR1 shares significant similarities with that of CXCR428, the only other chemokine receptor whose structure has been determined (Fig. 3; Supplementary Fig. 8). However, there are some notable differences reflecting the modifications made to the sequence of CXCR4 required for crystallization (insertion of T4 lysozyme in ICL3, removal of 33 C-terminal residues and L125W mutation), the amino acid sequence differences between the two proteins, the influence of the planar phospholipid bilayer, or the rotational diffusion of the protein.

Figure 3
Structural comparison of CXCR1 (cyan, PDB ID: 2LNL) and CXCR4 (pink, PDB ID: 3ODU)

The CXCR1 helices are well defined by the spectroscopic data. For example, a plot of the measured values of the amide 1H-15N DC versus residue number yields a characteristic wave-like pattern29 reflecting helical structure (Fig. 1d), with breaks in the waves corresponding to helix termini or kinks. For example, the DC data show the presence of a kink that changes the direction of helix TM2 (residues 74-101) at Phe88, coinciding with a kink at the same location in CXCR4 (Fig. 3). The extracellular start of TM7, just following ECL3, is tilted towards the receptor’s central axis in CXCR1, although it is less well defined in CXCR1 than in CXCR4 where residues in the N-terminus of TM7 interact with the added compound IT1t28. Helix TM7 is also about one turn longer at the intracellular end than its counterpart in CXCR4, extending to Ile308, three residues beyond the conserved GPCR sequence NPxxY. Furthermore, residues immediately preceding the mobile C terminus of CXCR1 form a well-defined helix (H8; residues Gln310 to Ala321) that is absent in the structure of CXCR4 (Fig. 2, ,3;3; Supplementary Fig. 8). H8 has a distinctly amphipathic amino acid sequence and aligns along the membrane surface, indicating that the phospholipid bilayer may play a role in stabilizing its conformation.

Figure 2
Three-dimensional structure of CXCR1

The NMR data further reveal the presence of two disulfide bonds (Fig. 2b; Supplementary Fig. 9) that are also present in CXCR4, one connecting the N-terminus to the extracellular start of TM7 (Cys30-Cys277) and the other connecting the extracellular end of TM3 to ECL2 (Cys110-Cys187). These Cys pairs are highly conserved in the sequences of chemokine receptors and are important for ligand binding. Together, they play a significant role in shaping the extracellular structure of the receptor, and also provide useful restraints for structure determination. The long ECL2 of CXCR1 forms a β-hairpin whose structure is constrained by the Cys110-Cys187 disulfide bond. A similar structure is observed in CXCR4 and in many other GPCRs, despite a lack of amino acid sequence similarity in this region. However, the ECL2 β-hairpin of CXCR1 is less well defined than that of CXCR4, consistent with the presence of two Pro residues in CXCR1.

In both CXCR1 and CXCR4, charged residues are mainly located near the membrane-water interface (Supplementary Fig. 10), with negative charges clustered in the extracellular loops where they can play a role in ligand binding and receptor activation. In addition, four charged residues, contributed by helices TM2 (Asp85), TM3 (Lys117) and TM7 (Asp288, Glu291), form a polar cluster in the core of the helical bundle of CXCR1 that may have important consequences for ligand binding and receptor signal transduction. One of these residues (Asp288) is not conserved in CXCR4 and may contribute to the differences in biological activities of the two chemokine receptors.

The intracellular loops of GPCRs are critical for G-protein interactions11. Modification of ICL3 by insertion of T4 lysozyme between TM5 and TM6, rendered CXCR4 incapable of activating G-proteins28. In contrast, unmodified CXCR1 is fully active with respect to both G-protein activation and chemokine binding, and its three intracellular loops are structurally well defined (Fig. 2, Fig. 3c, Supplementary Fig. 8). Notably, ICL3, which is important for CXCR1 coupling to G-proteins and involved in calcium mobilization, chemokine-mediated migration, and cell adhesion, extends from Thr228 to Gln236, connecting helices TM5 and TM6, which are both one turn shorter than the corresponding helices in CXCR4. ICL3 protrudes into the cytoplasm where it is available for G-protein binding. Its sequence shares significant homology with other GPCRs; therefore, the ability to observe the structure of an intact GPCR provides an opportunity to propose structure-based mechanisms of G-protein binding and activation.

CXCR1 has been the subject of significant molecular modeling efforts aimed at understanding its interactions with small molecule inhibitors, including compounds active in reducing breast cancer metastasis30. The structure of CXCR1 determined in a lipid bilayer membrane should facilitate these studies. The solid-state NMR approach used for structure determination has several significant advantages. The protein resides in fully hydrated liquid crystalline phospholipid bilayers at physiological conditions of temperature and pH, and no detergents or non-native lipid phases are present. The protein sequence is unmodified, with no truncations, mutations, or insertions of foreign proteins. The phospholipid composition can be varied, and other components, such as cholesterol, can be added. Other proteins or small molecules, including chemokines, G-proteins, nanobodies, and drugs can be added directly to the samples, enabling detection of any structural changes by direct spectroscopic and structural comparisons. NMR is adept at describing both overall and local protein dynamics. Measurements of DCs and isotropic chemical shifts to provide molecular orientation and dihedral angle restraints, combined with refinement in a membrane environment, facilitate structure determination by molecular fragment replacement. These can be supplemented with orientation restraints derived from rotationally averaged chemical shift anisotropy powder patterns to further improve structural accuracy and precision. Thus, we anticipate that this method will enable structure determination and structure-activity studies of other GPCRs as well as a wide range of other membrane proteins under near-native conditions.


Sample Preparation

Full-length human CXCR1 (residues 1 to 350) was expressed with an N-terminal GST partner and a C-terminal His6 tag in E. coli BL21 cells. Isotopically labeled samples were obtained by growing bacteria in M9 media containing 15N labeled ammonium sulfate and 13C6-glucose or 2-13C-glycerol (Cambridge Isotope Laboratories). A sample of selectively 13C/15N-Phe labeled CXCR1 was also prepared. After cell lysis, the GST-CXCR1-His6 fusion protein was bound to Ni-NTA resin. CXCR1 was separated from GST by incubation with thrombin and then purified and refolded in DMPC proteoliposomes by detergent dialysis22,23,31. The resulting proteoliposomes were suspended in buffer, isolated by ultra-centrifugation, and packed as a hydrated pellet into the MAS rotor. Detailed sample preparation methods are provided in Supplementary Information.

Ligand binding and G-protein activation

To assay IL-8 ligand binding, CXCR1 proteoliposomes were incubated with varying concentrations of 125I-labeled and unlabeled IL-8, and bound IL-8 was determined by measuring radioactivity in a scintillation counter after removing any free IL-8 ligand (Supplementary Fig. 1a)22,31. To assay G-protein activation, CXCR1 proteoliposomes were reconstituted with Gi/o protein trimer, and used to measure 35S-GTPγS binding as a function of agonist IL-8 concentration (Supplementary Fig. 1b)22,31. Refolded CXCR1 binds IL-8 (Kd ~ 1–5 nM) and activates G-protein in a ligand-dependent manner (EC50 ~ 1 nM), with affinities similar to those reported in the literature1,32.

NMR Spectroscopy

NMR experiments, experimental parameters and measurements of restraints are described in Supplementary Information, including Supplementary Table 2. 13C chemical shifts were externally referenced to DSS by setting the adamantane methylene carbons to a 13C chemical shift frequency of 40.48 ppm; 15N chemical shifts were externally referenced to liquid ammonia by setting the ammonium sulfate nitrogen to 26.8 ppm33,34. Fast rotational diffusion (>105 Hz) of the protein was verified by analysis of 13CO powder pattern line shapes35. Sample integrity was ascertained by monitoring one- and two-dimensional spectra.

Resonances from residues 20-325 of CXCR1 were all assigned, except for those corresponding to seven Pro residues (P22, P93, P170, P180, P185, P214 and P257) and one Arg (R285). Two disulfide bonds (C30-C277, C110-C187) were determined from the characteristic CB and CA chemical shifts that reflect the oxidation states of Cys sites36. Backbone dihedral angle (ϕ, ψ) restraints were derived from the experimentally measured isotropic chemical shifts using CS-Rosetta20 and TALOS37,38. Values of the experimental 1H-15N DC and 1H-13CA DC used in the structure calculations were measured from the perpendicular edge frequencies of the respective rotationally averaged powder patterns. For each DC, the perpendicular edge frequency was multiplied by 4 to obtain the frequency of dipolar splitting between the parallel edges of the Pake doublet. In most measurements, the sign of the 1H-15N DC could be determined unambiguously39. In cases where this was not possible, and for all of the 1H-13CA DC data, the DC restraints were implemented as absolute values in the structure calculations.

Structure Calculations

Structure calculations were performed in three stages using the programs: CS-Rosetta20; Rosetta19,21 (including both the coarse-grained and all-atom potentials19 as well as the implicit membrane potential21 available in Rosetta version 3.2); and Xplor-NIH26.

In the first stage, we used CS-Rosetta together with the amino acid sequence of CXCR1 and the assigned isotropic chemical shifts from CA, CB, CO, and N protein sites, to generate a molecular fragment database, containing 67,784 9-residue fragments and 69,204 3-residue fragments.

In the second stage, we used the resulting molecular fragment database, together with the experimental DC restraints and the structure of rhodopsin (PDB ID: 1F88)25 as topology template, to fold 20,000 structural models with the coarse-grained and implicit membrane potentials of Rosetta. These coarse-grained structural models were evaluated according to their Rosetta energy and backbone CA RMSD to the lowest energy structure. The 1,000 lowest energy models were selected for further refinement using the all-atom energy function and implicit membrane environment of Rosetta, together with the experimental DC restraints, and the experimentally determined disulfide bond restraints. The Rosetta all-atom relax protocol was implemented for 10 cycles with an increasing 1H-15N DC restraint weighting factor ramped from 1 to 3 kcal•mol-1•Hz-2. The lowest energy structure was selected for further refinement in the next stage. Convergence of the Rosetta calculations is shown in Supplementary Fig. 11.

In the third and final stage, structure refinement was performed using a simulated annealing protocol with Xplor-NIH internal variable molecular dynamics40 and all the experimental NMR restraints. During simulated annealing, the temperature was lowered from 500 K to 50 K. Experimentally determined disulfide bonds were included by explicit definition in the molecular structure file of CXCR1. Backbone dihedral angles were imposed with a range of ±2° for helices and ±30° for loops, and a fixed force constant (1,000 kcal•mol-1•rad-2). 1H-15N DC restraints were imposed with a range of ±2 kHz and a ramped force constant (0.1-2.5 kcal•mol-1•kHz-2). 1H-13CA DC restraints were imposed with a range of ±4 kHz and a ramped force constant (0.05-1.25 kcal•mol-1•kHz-2). The protocol also included a potential for knowledge-based torsion angles41 implemented with a dimensionless force constant (0.2), a potential for the radius of gyration42 implemented for residues 28 to 325 with a fixed force constant (10 kcal•mol-1•Å-2), plus energy terms to enforce covalent geometry and prevent atomic overlap43.

The magnitude and symmetry of the molecular alignment tensor were fixed, with values of the axial alignment (Da) and rhombicity (Rh) parameters set to 10.52 kHz and 0, respectively, as expected for a membrane protein in phospholipid bilayers with an order parameter of 1.0 and an amide NH bond length of 1.05 Å. The 1H-13CA DC alignment tensor was normalized to the maximum value of the 1H-15N DC.

A total of 100 structures were calculated and the 10 lowest energy structures were selected as the structural ensemble for analysis (Fig. 1e, Supplementary Table 1). Ramachandran Plot Statistics were evaluated with the program PROCHECK44. Molecular structures were analyzed and visualized with Pymol45. RMSDs and R factors, reflecting correlations between experimentally observed values of the restraints and values calculated from the refined structure, were estimated as described46.

Supplementary Material


This research was supported by grants R01EB005161, R01GM075877, R21GM94727, R21GM075917, P01AI074805 and P41EB002031 from the National Institutes of Health. Additional support came from Cambridge Isotope Laboratories. F.C. was supported by fellowships from the Swiss National Science Foundation (PBBSP3-123151) and the Novartis Foundation.


Author Contributions S.J.O. designed the study. S.P. optimized CXCR1 purification, refolding and NMR sample preparation. B.B.D. performed the NMR experiments. H.J.N. assisted in NMR data analysis. H.K. developed initial protocols for CXCR1 purification, refolding and functional assays. K.M. assisted in the revision of these methods for NMR experiments. A.D.D. tested samples for their suitability for NMR experiments. F.C., M.C., K.M. expressed and purified CXCR1. F.M.M. and Y. T. performed the structure calculations. S.J.O., S.P., B.D., F.M.M. and Y.T. prepared the figures and wrote the paper.

Atomic coordinates for residues 29 to 324 of CXCR1 and NMR restraints have been deposited in the Protein Data Bank (PDB ID: 2LNL). Assigned NMR frequencies have been deposited in the Biological Magnetic Resonance Bank (BMRB code: 18170).

The authors declare no competing financial interests.

Supplementary information is linked to the online version of the paper at


1. Holmes WE, Lee J, Kuang WJ, Rice GC, Wood WI. Structure and functional expression of a human interleukin-8 receptor. Science. 1991;253:1278–1280. [PubMed]
2. Sallusto F, Baggiolini M. Chemokines and leukocyte traffic. Nat Immunol. 2008;9:949–952. [PubMed]
3. Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res. 2008;14:6735–6741. [PubMed]
4. Rajagopal S, Rajagopal K, Lefkowitz RJ. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov. 2010;9:373–386. [PMC free article] [PubMed]
5. Goncalves JA, Ahuja S, Erfani S, Eilers M, Smith SO. Structure and function of G protein-coupled receptors using NMR spectroscopy. Prog Nucl Magn Reson Spectrosc. 2010;57:159–180. [PMC free article] [PubMed]
6. Rosenbaum DM, Rasmussen SG, Kobilka BK. The structure and function of G-protein-coupled receptors. Nature. 2009;459:356–363. [PMC free article] [PubMed]
7. Katritch V, Cherezov V, Stevens RC. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol Sci. 2012;33:17–27. [PMC free article] [PubMed]
8. Rosenbaum DM, et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science. 2007;318:1266–1273. [PubMed]
9. Rasmussen SG, et al. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature. 2007;450:383–387. [PubMed]
10. Landau EM, Rosenbusch JP. Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc Natl Acad Sci U S A. 1996;93:14532–14535. [PubMed]
11. Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nature reviews. 2008;9:60–71. [PubMed]
12. Crump MP, et al. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J. 1997;16:6996–7007. [PubMed]
13. Rajagopalan L, Rajarathnam K. Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain. J Biol Chem. 2004;279:30000–30008. [PubMed]
14. Park SH, Casagrande F, Cho L, Albrecht L, Opella SJ. Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy. J Mol Biol. 2011;414:194–203. [PMC free article] [PubMed]
15. Das BB, et al. Structure Determination of a Membrane Protein in Proteoliposomes. J Am Chem Soc. 2012;134:2047–2056. [PMC free article] [PubMed]
16. McDermott A. Structure and dynamics of membrane proteins by magic angle spinning solid-state NMR. Annu Rev Biophys. 2009;38:385–403. [PubMed]
17. Opella SJ, Marassi FM. Structure determination of membrane proteins by NMR spectroscopy. Chem Rev. 2004;104:3587–3606. [PMC free article] [PubMed]
18. Edidin M. Rotational and translational diffusion in membranes. Annu Rev Biophys Bioeng. 1974;3:179–201. [PubMed]
19. Das R, Baker D. Macromolecular modeling with rosetta. Annu Rev Biochem. 2008;77:363–382. [PubMed]
20. Shen Y, et al. Consistent blind protein structure generation from NMR chemical shift data. Proc Natl Acad Sci U S A. 2008;105:4685–4690. [PubMed]
21. Yarov-Yarovoy V, Schonbrun J, Baker D. Multipass membrane protein structure prediction using Rosetta. Proteins. 2006;62:1010–1025. [PMC free article] [PubMed]
22. Park SH, et al. High-resolution NMR spectroscopy of a GPCR in aligned bicelles. J Am Chem Soc. 2006;128:7402–7403. [PMC free article] [PubMed]
23. Park SH, et al. Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination. Biochim Biophys Acta. 2012;1818:584–591. [PMC free article] [PubMed]
24. Park SH, et al. Local and global dynamics of the G protein-coupled receptor CXCR1. Biochemistry. 2011;50:2371–2380. [PMC free article] [PubMed]
25. Palczewski K, et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science. 2000;289:739–745. [PubMed]
26. Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM. The Xplor-NIH NMR molecular structure determination package. J Magn Reson. 2003;160:65–73. [PubMed]
27. Krebs A, Edwards PC, Villa C, Li J, Schertler GF. The three-dimensional structure of bovine rhodopsin determined by electron cryomicroscopy. J Biol Chem. 2003;278:50217–50225. [PubMed]
28. Wu B, et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science. 2010;330:1066–1071. [PMC free article] [PubMed]
29. Mesleh MF, et al. Dipolar waves map the structure and topology of helices in membrane proteins. J Am Chem Soc. 2003;125:8928–8935. [PMC free article] [PubMed]
30. Ginestier C, et al. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J Clin Invest. 2010;120:485–497. [PMC free article] [PubMed]
31. Casagrande F, Maier K, Kiefer H, Opella SJ, Park SH. Production of Membrane Proteins. Wiley-VCH Verlag GmbH & Co. KGaA; 2011. pp. 297–316.
32. Murphy PM, Tiffany HL. Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science. 1991;253:1280–1283. [PubMed]
33. Wishart DS, et al. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR. 1995;6:135–140. [PubMed]
34. Morcombe CR, Zilm KW. Chemical shift referencing in MAS solid state NMR. J Magn Reson. 2003;162:479–486. [PubMed]
35. Park SH, Das BB, De Angelis AA, Scrima M, Opella SJ. Mechanically, magnetically, and “rotationally aligned” membrane proteins in phospholipid bilayers give equivalent angular constraints for NMR structure determination. J Phys Chem B. 2010;114:13995–14003. [PMC free article] [PubMed]
36. Sharma D, Rajarathnam K. 13C NMR chemical shifts can predict disulfide bond formation. J Biomol NMR. 2000;18:165–171. [PubMed]
37. Cornilescu G, Delaglio F, Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR. 1999;13:289–302. [PubMed]
38. Shen Y, Delaglio F, Cornilescu G, Bax A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR. 2009;44:213–223. [PMC free article] [PubMed]
39. Denny JK, Wang J, Cross TA, Quine JR. PISEMA powder patterns and PISA wheels. J Magn Reson. 2001;152:217–226. [PubMed]
40. Schwieters CD, Clore GM. Internal coordinates for molecular dynamics and minimization in structure determination and refinement. J Magn Reson. 2001;152:288–302. [PubMed]
41. Kuszewski J, Gronenborn AM, Clore GM. Improvements and extensions in the conformational database potential for the refinement of NMR and X-ray structures of proteins and nucleic acids. J Magn Reson. 1997;125:171–177. [PubMed]
42. Kuszewski J, Gronenborn AM, Clore GM. Improving the Packing and Accuracy of NMR Structures with a Pseudopotential for the Radius of Gyration. J Am Chem Soc. 1999;121:2337–2338.
43. Nilges M, Clore GM, Gronenborn AM. Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms. Circumventing problems associated with folding. FEBS Lett. 1988;239:129–136. [PubMed]
44. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR. 1996;8:477–486. [PubMed]
45. DeLano WL. PyMol. 2005
46. Clore GM, Garrett DS. R-factor, Free R, and Complete Cross-Validation for Dipolar Coupling Refinement of NMR Structures. J Am Chem Soc. 1999;121:9008–9012.