Journal of Biomolecular NMR

, 51:227 | Cite as

High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data

  • Ming Tang
  • Lindsay J. Sperling
  • Deborah A. Berthold
  • Charles D. Schwieters
  • Anna E. Nesbitt
  • Andrew J. Nieuwkoop
  • Robert B. Gennis
  • Chad M. Rienstra


X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) are the staple methods for revealing atomic structures of proteins. Since crystals of biomolecular assemblies and membrane proteins often diffract weakly and such large systems encroach upon the molecular tumbling limit of solution NMR, new methods are essential to extend structures of such systems to high resolution. Here we present a method that incorporates solid-state NMR restraints alongside of X-ray reflections to the conventional model building and refinement steps of structure calculations. Using the 3.7 Å crystal structure of the integral membrane protein complex DsbB-DsbA as a test case yielded a significantly improved backbone precision of 0.92 Å in the transmembrane region, a 58% enhancement from using X-ray reflections alone. Furthermore, addition of solid-state NMR restraints greatly improved the overall quality of the structure by promoting 22% of DsbB transmembrane residues into the most favored regions of Ramachandran space in comparison to the crystal structure. This method is widely applicable to any protein system where X-ray data are available, and is particularly useful for the study of weakly diffracting crystals.


Membrane protein Solid-state NMR X-ray reflections High resolution Joint calculation 

Supplementary material

10858_2011_9565_MOESM1_ESM.pdf (444 kb)
Supplementary material 1 Experimental procedures, 13C-13C correlation spectra of glycerol labeled DsbB samples and validations of structural calculations of DsbB-DsbA. (PDF 443 kb)


  1. Bayrhuber M, Meins T, Habeck M, Becker S, Giller K, Villinger S, Vonrhein C, Griesinger C, Zweckstetter M, Zeth K (2008) Structure of the human voltage-dependent anion channel. Proc Natl Acad Sci USA 105:15370–15375ADSCrossRefGoogle Scholar
  2. Cady SD, Schmidt-Rohr K, Wang J, Soto CS, DeGrado WF, Hong M (2010) Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463:689–692ADSCrossRefGoogle Scholar
  3. Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid-state magic-angle- spinning NMR spectroscopy. Nature 420:98–102ADSCrossRefGoogle Scholar
  4. Chen YW, Clore GM (2000) A systematic case study on using NMR models for molecular replacement: p53 tetramerization domain revisited. Acta Crystallogr D Biol Crystallogr 56:1535–1540CrossRefGoogle Scholar
  5. Gabel F, Simon B, Nilges M, Petoukhov M, Svergun D, Sattler M (2008) A structure refinement protocol combining NMR residual dipolar couplings and small angle scattering restraints. J Biomol NMR 41:199–208CrossRefGoogle Scholar
  6. Gautier A, Mott HR, Bostock MJ, Kirkpatrick JP, Nietlispach D (2010) Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nat Struct Mol Biol 17:768–774CrossRefGoogle Scholar
  7. Grishaev A, Wu J, Trewhella J, Bax A (2005) Refinement of multidomain protein structures by combination of solution small-angle X-ray scattering and NMR data. J Am Chem Soc 127:16621–16628CrossRefGoogle Scholar
  8. Grishaev A, Tugarinov V, Kay LE, Trewhella J, Bax A (2008a) Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints. J Biomol NMR 40:95–106CrossRefGoogle Scholar
  9. Grishaev A, Ying J, Canny MD, Pardi A, Bax A (2008b) Solution structure of tRNAVal from refinement of homology model against residual dipolar coupling and SAXS data. J Biomol NMR 42:99–109CrossRefGoogle Scholar
  10. Harper JK, Grant DM, Zhang Y, Lee PL, Von Dreele R (2006) Characterizing challenging microcrystalline solids with solid-state NMR shift tensor and synchrotron X-ray powder diffraction data: structural analysis of ambuic acid. J Am Chem Soc 128:1547–1552CrossRefGoogle Scholar
  11. Hiller S, Garces RG, Malia TJ, Orekhov VY, Colombini M, Wagner G (2008) Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321:1206–1210ADSCrossRefGoogle Scholar
  12. Hu F, Luo W, Hong M (2010) Mechanisms of proton conduction and gating in influenza M2 proton channels from solid-state NMR. Science 330:505–508ADSCrossRefGoogle Scholar
  13. Inaba K, Murakami S, Suzuki M, Nakagawa A, Yamashita E, Okada K, Ito K (2006) Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation. Cell 127:789–801CrossRefGoogle Scholar
  14. Inaba K, Murakami S, Nakagawa A, Iida H, Kinjo M, Ito K, Suzuki M (2009) Dynamic nature of disulphide bond formation catalysts revealed by crystal structures of DsbB. EMBO J 28:779–791CrossRefGoogle Scholar
  15. Ito K, Inaba K (2008) The disulfide bond formation (Dsb) system. Curr Opin Struct Biol 18:450–458CrossRefGoogle Scholar
  16. Jehle S, Rajagopal P, Bardiaux B, Markovic S, Kuhne R, Stout JR, Higman VA, Klevit RE, van Rossum BJ, Oschkinat H (2010) Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers. Nat Struct Mol Biol 17:1037–1042CrossRefGoogle Scholar
  17. Koharudin LM, Furey W, Liu H, Liu YJ, Gronenborn AM (2009) The phox domain of sorting nexin 5 lacks phosphatidylinositol 3-phosphate (PtdIns(3)P) specificity and preferentially binds to phosphatidylinositol 4, 5-bisphosphate (PtdIns(4, 5)P2). J Biol Chem 284:23697–23707CrossRefGoogle Scholar
  18. Kuszewski J, Schwieters CD, Garrett DS, Byrd RA, Tjandra N, Clore GM (2004) Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments. J Am Chem Soc 126:6258–6273CrossRefGoogle Scholar
  19. Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) Procheck—a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  20. Li Y, Berthold DA, Gennis RB, Rienstra CM (2008) Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy. Protein Sci 17:199–204CrossRefGoogle Scholar
  21. Li W, Schulman S, Dutton RJ, Boyd D, Beckwith J, Rapoport TA (2010) Structure of a bacterial homologue of vitamin K epoxide reductase. Nature 463:507–512ADSCrossRefGoogle Scholar
  22. Liu L, Koharudin LM, Gronenborn AM, Bahar I (2009) A comparative analysis of the equilibrium dynamics of a designed protein inferred from NMR, X-ray, and computations. Proteins 77:927–939CrossRefGoogle Scholar
  23. Mahalakshmi R, Marassi FM (2008) Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-state NMR. Biochemistry 47:6531–6538CrossRefGoogle Scholar
  24. Malojčić G, Owen RL, Grimshaw JPA, Glockshuber R (2008) Preparation and structure of the charge-transfer intermediate of the transmembrane redox catalyst DsbB. FEBS Lett 582:3301–3307CrossRefGoogle Scholar
  25. Maly T, Debelouchina GT, Bajaj VS, Hu KN, Joo CG, Mak-Jurkauskas ML, Sirigiri JR, van der Wel PCA, Herzfeld J, Temkin RJ, Griffin RG (2008) Dynamic nuclear polarization at high magnetic fields. J Chem Phys 128:052211ADSCrossRefGoogle Scholar
  26. Matei E, Furey W, Gronenborn AM (2008) Solution and crystal structures of a sugar binding site mutant of cyanovirin-N: no evidence of domain swapping. Structure 16:1183–1194CrossRefGoogle Scholar
  27. McDermott A (2009) Structure and dynamics of membrane proteins by magic angle spinning solid-state NMR. Annu Rev Biophys 38:385–403MathSciNetCrossRefGoogle Scholar
  28. Nieuwkoop AJ, Rienstra CM (2010) Supramolecular protein structure determination by site-specific long-range intermolecular solid state NMR spectroscopy. J Am Chem Soc 132:7570–7571CrossRefGoogle Scholar
  29. Schroder GF, Levitt M, Brunger AT (2010) Super-resolution biomolecular crystallography with low-resolution data. Nature 464:1218–1222ADSCrossRefGoogle Scholar
  30. Schwieters CD, Clore GM (2007) A physical picture of atomic motions within the Dickerson DNA dodecamer in solution derived from joint ensemble refinement against NMR and large-angle X-ray scattering data. Biochemistry 46:1152–1166CrossRefGoogle Scholar
  31. Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160:65–73ADSCrossRefGoogle Scholar
  32. Schwieters CD, Suh JY, Grishaev A, Ghirlando R, Takayama Y, Clore GM (2010) Solution structure of the 128 kDa enzyme I dimer from Escherichia coli and its 146 kDa complex with HPr using residual dipolar couplings and small- and wide-angle X-ray scattering. J Am Chem Soc 132:13026–13045CrossRefGoogle Scholar
  33. Shaanan B, Gronenborn AM, Cohen GH, Gilliland GL, Veerapandian B, Davies DR, Clore GM (1992) Combining experimental information from crystal and solution studies—joint X-ray and NMR refinement. Science 257:961–964ADSCrossRefGoogle Scholar
  34. Sharma M, Yi M, Dong H, Qin H, Peterson E, Busath DD, Zhou HX, Cross TA (2010) Insight into the mechanism of the influenza a proton channel from a structure in a lipid bilayer. Science 330:509–512ADSCrossRefGoogle Scholar
  35. Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS plus : a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213–223CrossRefGoogle Scholar
  36. Shi L, Traaseth NJ, Verardi R, Cembran A, Gao J, Veglia G (2009) A refinement protocol to determine structure, topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints. J Biomol NMR 44:195–205CrossRefGoogle Scholar
  37. Sperling LJ, Berthold DA, Sasser TL, Jeisy-Scott V, Rienstra CM (2010) Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide bond forming enzyme DsbA. J Mol Biol 399:268–282CrossRefGoogle Scholar
  38. Tang M, Sperling LJ, Berthold DA, Nesbitt AE, Gennis RB, Rienstra CM (2011) Solid-state NMR study of the charge-transfer complex between ubiquinone-8 and disulfide bond generating membrane protein DsbB. J Am Chem Soc 133:4359–4366CrossRefGoogle Scholar
  39. Traaseth NJ, Shi L, Verardi R, Mullen DG, Barany G, Veglia G (2009) Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach. Proc Natl Acad Sci USA 106:10165–10170ADSCrossRefGoogle Scholar
  40. Van Horn WD, Kim HJ, Ellis CD, Hadziselimovic A, Sulistijo ES, Karra MD, Tian CL, Sonnichsen FD, Sanders CR (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324:1726–1729ADSCrossRefGoogle Scholar
  41. Verardi R, Shi L, Traaseth NJ, Walsh N, Veglia G (2011) Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method. Proc Natl Acad Sci USA 108:9101–9106ADSCrossRefGoogle Scholar
  42. Wang J, Zuo X, Yu P, Byeon IJ, Jung J, Wang X, Dyba M, Seifert S, Schwieters CD, Qin J, Gronenborn AM, Wang YX (2009) Determination of multicomponent protein structures in solution using global orientation and shape restraints. J Am Chem Soc 131:10507–10515CrossRefGoogle Scholar
  43. White SH (2009) Biophysical dissection of membrane proteins. Nature 459:344–346ADSCrossRefGoogle Scholar
  44. Wickramasinghe NP, Parthasarathy S, Jones CR, Bhardwaj C, Long F, Kotecha M, Mehboob S, Fung LWM, Past J, Samoson A, Ishii Y (2009) Nanomole-scale protein solid-state NMR by breaking intrinsic 1H T 1 boundaries. Nat Methods 6:215–218CrossRefGoogle Scholar
  45. Wylie BJ, Schwieters CD, Oldfield E, Rienstra CM (2009) Protein structure refinement using 13Cα chemical shift tensors. J Am Chem Soc 131:985–992CrossRefGoogle Scholar
  46. Zhou DH, Shea JJ, Nieuwkoop AJ, Franks WT, Wylie BJ, Mullen C, Sandoz D, Rienstra CM (2007) Solid-state protein-structure determination with proton-detected triple-resonance 3D magic-angle spinning NMR spectroscopy. Angew Chem Int Ed 46:8380–8383CrossRefGoogle Scholar
  47. Zhou YP, Cierpicki T, Jimenez RHF, Lukasik SM, Ellena JF, Cafiso DS, Kadokura H, Beckwith J, Bushweller JH (2008) NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol Cell 31:896–908CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Ming Tang
    • 1
  • Lindsay J. Sperling
    • 1
  • Deborah A. Berthold
    • 1
  • Charles D. Schwieters
    • 2
  • Anna E. Nesbitt
    • 1
  • Andrew J. Nieuwkoop
    • 1
  • Robert B. Gennis
    • 1
  • Chad M. Rienstra
    • 1
  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Division of Computational Bioscience, Center for Information TechnologyNational Institutes of HealthBethesdaUSA

Personalised recommendations