Journal of Biomolecular NMR

, Volume 43, Issue 2, pp 63–78 | Cite as

De novo protein structure generation from incomplete chemical shift assignments

  • Yang Shen
  • Robert Vernon
  • David Baker
  • Ad BaxEmail author


NMR chemical shifts provide important local structural information for proteins. Consistent structure generation from NMR chemical shift data has recently become feasible for proteins with sizes of up to 130 residues, and such structures are of a quality comparable to those obtained with the standard NMR protocol. This study investigates the influence of the completeness of chemical shift assignments on structures generated from chemical shifts. The Chemical-Shift-Rosetta (CS-Rosetta) protocol was used for de novo protein structure generation with various degrees of completeness of the chemical shift assignment, simulated by omission of entries in the experimental chemical shift data previously used for the initial demonstration of the CS-Rosetta approach. In addition, a new CS-Rosetta protocol is described that improves robustness of the method for proteins with missing or erroneous NMR chemical shift input data. This strategy, which uses traditional Rosetta for pre-filtering of the fragment selection process, is demonstrated for two paramagnetic proteins and also for two proteins with solid-state NMR chemical shift assignments.


NMR chemical shift Protein structure prediction Solid-state NMR structure determination Paramagnetic protein CS-Rosetta 



This work was funded by the Intramural Research Program of the NIDDK, NIH, and by the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH; the NIGMS, NIH, and the Howard Hughes Medical Institutes (to D. B.). We also thank Rosetta@home participants and the BOINC project for contributing computing power.

Supplementary material

10858_2008_9288_MOESM1_ESM.pdf (1.2 mb)
MOESM1 (PDF 1231 kb)


  1. Agarwal V, Diehl A, Skrynnikov N, Reif B (2006) High resolution H-1 detected H-1, C-13 correlation spectra in MAS solid-state NMR using deuterated proteins with selective H-1, H-2 isotopic labeling of methyl groups. J Am Chem Soc 128:12620–12621CrossRefGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  3. Ando I, Kameda T, Asakawa N, Kuroki S, Kurosu H (1998) Structure of peptides and polypeptides in the solid state as elucidated by NMR chemical shift. J Mol Struct 441:213–230CrossRefADSGoogle Scholar
  4. Andreini C, Bertini I, Rosato A (2004) A hint to search for metalloproteins in gene banks. Bioinformatics 20:1373–1380CrossRefGoogle Scholar
  5. Asakura T, Demura M, Date T, Miyashita N, Ogawa K, Williamson MP (1997) NMR study of silk I structure of Bombyx mori silk fibroin with N-15- and C-13-NMR chemical shift contour plots. Biopolymers 41:193–203CrossRefGoogle Scholar
  6. Barnwal RP, Rout AK, Chary KVR, Atreya HS (2008) Rapid measurement of pseudocontact shifts in paramagnetic proteins by GFT NMR spectroscopy. Open Magn Reson J 1:16–28CrossRefGoogle Scholar
  7. Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006) C-13-detected protonless NMR spectroscopy of proteins in solution. Prog Nucl Magn Reson Spectrosc 48:25–45CrossRefGoogle Scholar
  8. Bertini I, Luchinat C, Parigi G, Pierattelli R (2005) NMR spectroscopy of paramagnetic metalloproteins. Chembiochem 6:1536–1549CrossRefGoogle Scholar
  9. Bowers PM, Strauss CEM, Baker D (2000) De novo protein structure determination using sparse NMR data. J Biomol NMR 18:311–318CrossRefGoogle Scholar
  10. Case DA (1995) Calibration of ring-current effects in proteins and nucleic acids. J Biomol NMR 6:341–346CrossRefGoogle Scholar
  11. 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 420:98–102CrossRefADSGoogle Scholar
  12. Castellani F, van Rossum BJ, Diehl A, Rehbein K, Oschkinat H (2003) Determination of solid-state NMR structures of proteins by means of three-dimensional 15N–13C–13C dipolar correlation spectroscopy and chemical shift analysis. Biochemistry 42:11476–11483CrossRefGoogle Scholar
  13. Cavalli A, Salvatella X, Dobson CM, Vendruscolo M (2007) Protein structure determination from NMR chemical shifts. Proc Natl Acad Sci USA 104:9615–9620CrossRefADSGoogle Scholar
  14. Cavanagh J, Fairbrother WJ, Palmer AG, Rance M, Skelton NJ (2007) Protein NMR spectroscopy: principles and practice, 2nd edn. Academic Press, San Diego, CAGoogle Scholar
  15. Chevelkov V, Rehbein K, Diehl A, Reif B (2006) Ultrahigh resolution in proton solid-state NMR spectroscopy at high levels of deuteration. Angew Chem Int Ed 45:3878–3881CrossRefGoogle Scholar
  16. Cornilescu G, Marquardt JL, Ottiger M, Bax A (1998) Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J Am Chem Soc 120:6836–6837CrossRefGoogle Scholar
  17. Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13:289–302CrossRefGoogle Scholar
  18. Das R, Baker D (2008) Macromolecular modeling with Rosetta. Annu Rev Biochem 77:363–382CrossRefGoogle Scholar
  19. Delaglio F, Kontaxis G, Bax A (2000) Protein structure determination using Molecular Fragment Replacement and NMR dipolar couplings. J Am Chem Soc 122:2142–2143CrossRefGoogle Scholar
  20. Doreleijers JF, Nederveen AJ, Vranken W, Lin JD, Bonvin A, Kaptein R, Markley JL, Ulrich EL (2005) BioMagResBank databases DOCR and FRED containing converted and filtered sets of experimental NMR restraints and coordinates from over 500 protein PDB structures. J Biomol NMR 32:1–12CrossRefGoogle Scholar
  21. Gardner KH, Rosen MK, Kay LE (1997) Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR. Biochemistry 36:1389–1401CrossRefGoogle Scholar
  22. Gong HP, Shen Y, Rose GD (2007) Building native protein conformation from NMR backbone chemical shifts using Monte Carlo fragment assembly. Protein Sci 16:1515–1521CrossRefGoogle Scholar
  23. Gryk MR, Hoch JC (2008) Local knowledge helps determine protein structures. Proc Natl Acad Sci USA 105:4533–4534CrossRefADSGoogle Scholar
  24. Haigh CW, Mallion RB (1979) Ring current theories in nuclear magnetic resonance. Prog Nucl Magn Reson Spectrosc 13:303–344CrossRefGoogle Scholar
  25. Igumenova TI, McDermott AE, Zilm KW, Martin RW, Paulson EK, Wand AJ (2004) Assignments of carbon NMR resonances for microcrystalline ubiquitin. J Am Chem Soc 126:6720–6727CrossRefGoogle Scholar
  26. Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of larger proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29:4659–4667CrossRefGoogle Scholar
  27. Kontaxis G, Delaglio F, Bax A (2005) Molecular fragment replacement approach to protein structure determination by chemical shift and dipolar homology database mining. Meth Enzymol 394:42–78CrossRefGoogle Scholar
  28. Koradi R, Billeter M, Wuthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14:51–55CrossRefGoogle Scholar
  29. Loquet A, Bardiaux B, Gardiennet C, Blanchet C, Baldus M, Nilges M, Malliavin T, Boeckmann A (2008) 3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints. J Am Chem Soc 130:3579–3589CrossRefGoogle Scholar
  30. Manolikas T, Herrmann T, Meier BH (2008) Protein structure determination from C-13 spin-diffusion solid-state NMR spectroscopy. J Am Chem Soc 130:3959–3966CrossRefGoogle Scholar
  31. Markley JL, Bax A, Arata Y, Hilbers CW, Kaptein R, Sykes BD, Wright PE, Wuthrich K (1998) IUPAC-IUBMB-IUPAB inter-union task group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy. Pure Appl Chem 70:117–142CrossRefGoogle Scholar
  32. Montelione GT, Wagner G (1990) Conformation-independent sequential NMR connections in isotope-enriched polypeptides by 1H–13C–15N triple-resonance experiments. J Magn Reson 87:183–188Google Scholar
  33. Moseley HNB, Sahota G, Montelione GT (2004) Assignment validation software suite for the evaluation and presentation of protein resonance assignment data. J Biomol NMR 28:341–355CrossRefGoogle Scholar
  34. Muller J, Lugovskoy AA, Wagner G, Lippard SJ (2002) NMR structure of the [2Fe–2S] ferredoxin domain from soluble methane monooxygenase reductase and interaction with its hydroxylase. Biochemistry 41:42–51CrossRefGoogle Scholar
  35. Nadaud PS, Helmus JJ, Jaroniec CP (2007) 13C and 15N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy. Biomol NMR Assign 1:117–120CrossRefGoogle Scholar
  36. Neal S, Nip AM, Zhang HY, Wishart DS (2003) Rapid and accurate calculation of protein H-1, C-13 and N-15 chemical shifts. J Biomol NMR 26:215–240CrossRefGoogle Scholar
  37. Neal S, Berjanskii M, Zhang HY, Wishart DS (2006) Accurate prediction of protein torsion angles using chemical shifts and sequence homology. Magn Reson Chem 44:S158–S167CrossRefGoogle Scholar
  38. Pervushin K, Riek R, Wider G, Wuthrich K (1998) Transverse relaxation-optimized spectroscopy (TROSY) for NMR studies of aromatic spin systems in 13C-labeled proteins. J Am Chem Soc 120:6394–6400CrossRefGoogle Scholar
  39. Rohl CA, Strauss CEM, Misura KMS, Baker D (2004) Protein structure prediction using Rosetta. Meth Enzymol 383:66–93CrossRefGoogle Scholar
  40. Saito H (1986) Conformation-dependent C13 chemical shifts—a new means of conformational characterization as obtained by high resolution solid state C13 NMR. Magn Reson Chem 24:835–852CrossRefGoogle Scholar
  41. Shen Y, Bax A (2007) Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology. J Biomol NMR 38:289–302CrossRefGoogle Scholar
  42. Shen Y, Lange O, Delaglio F, Rossi P, Aramini JM, Liu GH, Eletsky A, Wu YB, Singarapu KK, Lemak A, Ignatchenko A, Arrowsmith CH, Szyperski T, Montelione GT, Baker D, Bax A (2008) Consistent blind protein structure generation from NMR chemical shift data. Proc Natl Acad Sci USA 105:4685–4690CrossRefADSGoogle Scholar
  43. Siemer AB, Ritter C, Ernst M, Riek R, Meier BH (2005) High-resolution solid-state NMR spectroscopy of the prion protein HET-s in its amyloid conformation. Angew Chem Int Ed 44:2441–2444CrossRefGoogle Scholar
  44. Spera S, Bax A (1991) Empirical correlation between protein backbone conformation and Cα and Cβ 13C nuclear magnetic resonance chemical shifts. J Am Chem Soc 113:5490–5492CrossRefGoogle Scholar
  45. Svensson LA, Thulin E, Forsen S (1992) Proline cis-trans isomers in calbindin D9K observed by X-ray crystallography. J Mol Biol 223:601–606CrossRefGoogle Scholar
  46. Tycko R (1996) Prospects for resonance assignments in multidimensional solid-state NMR spectra of uniformly labeled proteins. J Biomol NMR 8:239–251CrossRefGoogle Scholar
  47. Ulmer TS, Ramirez BE, Delaglio F, Bax A (2003) Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy. J Am Chem Soc 125:9179–9191CrossRefGoogle Scholar
  48. Venters RA, Farmer BT, Fierke CA, Spicer LD (1996) Characterizing the use of perdeuteration in NMR studies of large proteins C-13, N-15 and H-1 assignments of human carbonic anhydrase II. J Mol Biol 264:1101–1116CrossRefGoogle Scholar
  49. Wagner G, Pardi A, Wuthrich K (1983) Hydrogen-bond length and H-1-NMR chemical-shifts in proteins. J Am Chem Soc 105:5948–5949CrossRefGoogle Scholar
  50. Wang LY, Eghbalnia HR, Bahrami A, Markley JL (2005) Linear analysis of carbon-13 chemical shift differences and its application to the detection and correction of errors in referencing and spin system identifications. J Biomol NMR 32:13–22CrossRefGoogle Scholar
  51. Williamson MP, Asakura T (1993) Empirical comparisons of models for chemical-shift calculation in proteins. J Magn Reson B 101:63–71CrossRefGoogle Scholar
  52. Williamson MP, Kikuchi J, Asakura T (1995) Application of H1 NMR chemical shifts to measure the quality of protein structures. J Mol Biol 247:541–546Google Scholar
  53. Wishart DS, Sykes BD, Richards FM (1991) Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J Mol Biol 222:311–333CrossRefGoogle Scholar
  54. Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81CrossRefGoogle Scholar
  55. Wishart DS, Arndt D, Berjanskii M, Tang P, Zhou J, Lin G (2008) CS23D: a web server for rapid protein structure generation using NMR chemical shifts and sequence data. Nucleic Acids Res 36:496–502CrossRefGoogle Scholar
  56. Zech SG, Wand AJ, McDermott AE (2005) Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin. J Am Chem Soc 127:8618–8626CrossRefGoogle Scholar

Copyright information

© US Government 2008

Authors and Affiliations

  1. 1.Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaUSA
  2. 2.Department of Biochemistry and Howard Hughes Medical InstituteUniversity of WashingtonSeattleUSA

Personalised recommendations