A structure refinement protocol combining NMR residual dipolar couplings and small angle scattering restraints

  • F. Gabel
  • B. Simon
  • M. Nilges
  • M. Petoukhov
  • D. Svergun
  • M. SattlerEmail author


We present the implementation of a target function based on Small Angle Scattering data (Gabel et al. Eur Biophys J 35(4):313–327, 2006) into the Crystallography and NMR Systems (CNS) and demonstrate its utility in NMR structure calculations by simultaneous application of small angle scattering (SAS) and residual dipolar coupling (RDC) restraints. The efficiency and stability of the approach are demonstrated by reconstructing the structure of a two domain region of the 31 kDa nuclear export factor TAP (TIP-associated protein). Starting with the high resolution X-ray structures of the two individual TAP domains, the translational and orientational domain arrangement is refined simultaneously. We tested the stability of the protocol against variations of the SAS target parameters and the number of RDCs and their uncertainties. The activation of SAS restraints results in an improved translational clustering of the domain positions and lifts part of the fourfold degeneracy of their orientations (associated with a single alignment tensor). The resulting ensemble of structures reflects the conformational space that is consistent with the experimental SAS and RDC data. The SAS target function is computationally very efficient. SAS restraints can be activated at different levels of precision and only a limited SAS angular range is required. When combined with additional data from chemical shift perturbation, paramagnetic relaxation enhancement or mutational analysis the SAS refinement is an efficient approach for defining the topology of multi-domain and/or multimeric biomolecular complexes in solution based on available high resolution structures (NMR or X-ray) of the individual domains.


Nuclear Magnetic Resonance Quaternary structure Residual dipolar couplings Rigid body modeling Small angle scattering Structural refinement 



Ambiguous restraints for iterative assignment


Crystallography and NMR systems


Leucine rich repeat


Molecular dynamics


Non-crystallography symmetry


Nuclear magnetic resonance


Nuclear Overhauser effect


Normalized spatial discrepancy


Protein data bank


Paramagnetic relaxation enhancement


Residual dipolar couplings


Root mean square displacement


RNA recognition motif


Simulated annealing


Small angle scattering


Small angle neutron scattering


Small angle X-ray scattering


TIP-associated protein



This work is supported by the EU-grant: 3D repertoire, contract LSHG-CT-2005-512028 and the EU STREP FSG-V-RNA. MVP and DIS acknowledge support from the EU design study SAXIER (contact RIDS No 011934).

Supplementary material


  1. Aliprandi P, Sizun C, Perez J, Mareuil F, Caputo S, Leroy JL, Odaert B, Laalami S, Uzan M, Bontems F (2008) S1 ribosomal protein functions in translation initiation and ribonuclease RegB activation are mediated by similar RNA/protein interactions. An NMR and SAXS analysis. J Biol Chem [Epub ahead of print]Google Scholar
  2. Battiste JL, Wagner G (2000) Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. Biochemistry 39:5355–5365CrossRefGoogle Scholar
  3. Bax A (2003) Weak alignment offers new NMR opportunities to study protein structure and dynamics. Protein Sci 12(1):1–16CrossRefGoogle Scholar
  4. Bernado P, Blanchard L, Timmins P, Marion D, Ruigrok RW, Blackledge M (2005) A structural model for unfolded proteins from residual dipolar couplings and small-angle X-ray scattering. Proc Natl Acad Sci USA 102(47):17002–17007CrossRefADSGoogle Scholar
  5. Bernado P, Mylonas E, Petoukhov MV, Blackledge M, Svergun DI (2007) Structural characterization of flexible proteins using small-angle X-ray scattering. J Am Chem Soc 129:5656–5664CrossRefGoogle Scholar
  6. Blackledge M (2005) Recent progress in the study of biomolecular structure and dynamics in solution from residual dipolar couplings. Prog Nucl Mag Res Spectrosc 46:23–61CrossRefGoogle Scholar
  7. Boczko EM, Brooks CL 3rd (1995) First-principles calculation of the folding free energy of a three-helix bundle protein. Science 269(5222):393–396CrossRefADSGoogle Scholar
  8. Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D 54:905–921CrossRefGoogle Scholar
  9. Choy WY, Mulder FA, Crowhurst KA, Muhandiram DR, Millett IS, Doniach S, Forman-Kay JD, Kay LE (2002) Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques. J Mol Biol 316:101–112CrossRefGoogle Scholar
  10. Clore GM, Schwieters CD (2003) Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N–1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics. J Am Chem Soc 125:2902–2912CrossRefGoogle Scholar
  11. 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
  12. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293CrossRefGoogle Scholar
  13. Dominguez C, Boelens R, Bonvin AM (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125(7):1731–1737CrossRefGoogle Scholar
  14. Gabel F, Simon B, Sattler M (2006) A target function for quaternary structural refinement from small angle scattering and NMR orientational restraints. Eur Biophys J 35(4):313–327CrossRefGoogle Scholar
  15. Goult BT, Rapley JD, Dart C, Kitmitto A, Grossmann JG, Leyland ML, Lian LY (2007) Small-angle X-ray scattering and NMR studies of the conformation of the PDZ region of SAP97 and its interactions with Kir2.1. Biochemistry 46:14117–14128CrossRefGoogle Scholar
  16. 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(47):16621–16628CrossRefGoogle Scholar
  17. Grishaev A, Tugarinov V, Kay LE, Trewhella J, Bax A (2008) 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
  18. Hünenberger PH, Mark AE, van Gunsteren WF (1995) Computational approaches to study protein unfolding: hen egg white lysozyme as a case study. Proteins 21(3):196–213CrossRefGoogle Scholar
  19. Koch MH, Vachette P, Svergun DI (2003) Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution. Q Rev Biophys 36(2):147–227CrossRefGoogle Scholar
  20. Kozin MB, Svergun DI (2001) Automated matching of high- and low-resolution structural models. J Appl Crystallogr 34:33–41CrossRefGoogle Scholar
  21. Kuszewski J, Gronenborn AM, Clore GM (1999) Improving the packing and accuracy of NMR structures with a pseudopotential for the radius of gyration. J Am Chem Soc 121:2337–2338CrossRefGoogle Scholar
  22. Liker E, Fernandez E, Izaurralde E, Conti E (2000) The structure of the mRNA export factor TAP reveals a cis arrangement of a non-canonical RRM domain and an LRR domain. EMBO J 19(21):5587–5598CrossRefGoogle Scholar
  23. Linge JP, Williams MA, Spronk CA, Bonvin AM, Nilges M (2003) Refinement of protein structures in explicit solvent. Proteins 50:496–506CrossRefGoogle Scholar
  24. Mackereth CD, Simon B, Sattler M (2005) Extending the size of protein-RNA complexes studied by Nuclear Magnetic Resonance Spectroscopy. Chembiochem 6:1578–1584CrossRefGoogle Scholar
  25. Mareuil F, Sizun C, Perez J, Schoenauer M, Lallemand JY, Bontems F (2007) A simple genetic algorithm for the optimization of multidomain protein homology models driven by NMR residual dipolar couplings and small angle X-ray scattering data. Eur Biophys J 37(1):95–104CrossRefGoogle Scholar
  26. Marino M, Zou P, Svergun D, Garcia P, Edlich C, Simon B, Wilmanns M, Muhle-Goll C, Mayans O (2006) The Ig doublet Z1Z2: a model system for the hybrid analysis of conformational dynamics in Ig tandems from titin. Structure 14(9):1437–1447CrossRefGoogle Scholar
  27. Mattinen ML, Paakkonen K, Ikonen T, Craven J, Drakenberg T, Serimaa R, Waltho J, Annila A (2002) Quaternary structure built from subunits combining NMR and small-angle X-ray scattering data. Biophys J 83(2):1177–1183CrossRefGoogle Scholar
  28. Neylon C (2008) Small angle neutron and X-ray scattering in structural biology: recent examples from the literature. Eur Biophys J [Epub ahead of print]Google Scholar
  29. Petoukhov MV, Svergun DI (2005) Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys J 89:1237–1250CrossRefGoogle Scholar
  30. Prestegard JH, al-Hashimi HM, Tolman JR (2000) NMR structures of biomolecules using field oriented media and residual dipolar couplings. Q Rev Biophys 33(4):371–424CrossRefGoogle Scholar
  31. Svergun DI, Koch MH (2002) Advances in structure analysis using small-angle scattering in solution. Curr Opin Struct Biol 12(5):654–660CrossRefGoogle Scholar
  32. Svergun DI, Barberato C, Koch MHJ (1995) CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst 28:768–773CrossRefGoogle Scholar
  33. Svergun DI, Richard S, Koch MH, Sayers Z, Kuprin S, Zaccai G (1998) Protein hydration in solution: experimental observation by X-ray and neutron scattering. Proc Natl Acad Sci USA 95(5):2267–2272CrossRefADSGoogle Scholar
  34. Tidow H, Melero R, Mylonas E, Freund SM, Grossmann JG, Carazo JM, Svergun DI, Valle M, Fersht AR (2007) Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex. Proc Natl Acad Sci USA 104:12324–12329CrossRefADSGoogle Scholar
  35. Timmins PA, Zaccai G (1988) Low resolution structures of biological complexes studied by neutron scattering. Eur Biophys J 15(5):257–268CrossRefGoogle Scholar
  36. Yuzawa S, Ogura K, Horiuchi M, Suzuki NN, Fujioka Y, Kataoka M, Sumimoto F, Inagaki H (2004) Solution structure of the tandem Src homology 3 domains of p47phox in an autoinhibited form. J Biol Chem 279(28):29752–29760CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • F. Gabel
    • 1
    • 2
  • B. Simon
    • 1
  • M. Nilges
    • 3
  • M. Petoukhov
    • 4
    • 5
  • D. Svergun
    • 4
    • 5
  • M. Sattler
    • 1
    • 6
    • 7
    Email author
  1. 1.Structural and Computational Biology UnitEMBLHeidelbergGermany
  2. 2.Institut de Biologie StructuraleIBS Jean-Pierre Ebel, CEA-CNRS-UJFGrenoble CedexFrance
  3. 3.Unité de Bioinformatique StructuraleInstitut PasteurParisFrance
  4. 4.EMBL, Hamburg OutstationHamburgGermany
  5. 5.Institute of CrystallographyRussian Academy of SciencesMoscowRussia
  6. 6.Institute of Structural BiologyHelmholtz Zentrum MünchenNeuherbergGermany
  7. 7.Munich Center for Integrated Protein Science, Department ChemieTechnische Universität MünchenGarchingGermany

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