Harnessing the Combined Power of SAXS and NMR

  • A. M. Gronenborn
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1105)


Single types of methodologies are no longer sufficient to adequately describe complex biological structures. As a result, integrated approaches that combine complementary data are being developed. This chapter describes the integration of nuclear magnetic resonance and small-angle scattering approaches to characterize solution structures of multi-domain proteins.


Integrated structural biology Multi-domain proteins NMR SAXS Molecular dynamics simulations 



I thank all former and present members of Gronenborn laboratory for their contributions to our studies referenced here and Teresa Brosenitsch for excellent editorial help. This work was supported by a National Institutes of Health Grant RO1GM080642 (A.M.G.).


  1. 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(17):5656–5664. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bertini I, Luchinat C (1999) New applications of paramagnetic NMR in chemical biology. Curr Opin Chem Biol 3(2):145–151. CrossRefPubMedGoogle Scholar
  3. Bertini I, Ferella L, Luchinat C, Parigi G, Petoukhov MV, Ravera E, Rosato A, Svergun DI (2012) MaxOcc: a web portal for maximum occurrence analysis. J Biomol NMR 53(4):271–280. CrossRefPubMedGoogle Scholar
  4. Bhandari YR, Jiang W, Stahlberg EA, Stagno JR, Wang YX (2016) Modeling RNA topological structures using small angle X-ray scattering. Methods 103:18–24. CrossRefPubMedGoogle Scholar
  5. Cohen SL, Chait BT (2001) Mass spectrometry as a tool for protein crystallography. Annu Rev Biophys Biomol Struct 30:67–85. CrossRefPubMedGoogle Scholar
  6. Cowieson NP, Miles AJ, Robin G, Forwood JK, Kobe B, Martin JL, Wallace BA (2008) Evaluating protein: protein complex formation using synchrotron radiation circular dichroism spectroscopy. Proteins 70(4):1142–1146. CrossRefPubMedGoogle Scholar
  7. Debiec KT, Whitley MJ, Koharudin LMI, Chong LT, Gronenborn AM (2018) Integrating NMR/SAXS experiments and atomistic simulations – structure and dynamics of a two-domain protein. Biophys J 114(4):839–855. CrossRefGoogle Scholar
  8. Doniach S (2001) Changes in biomolecular conformation seen by small angle X-ray scattering. Chem Rev 101(6):1763–1778CrossRefGoogle Scholar
  9. Doniach S, Lipfert J (2012) Small and wide angle X-ray scattering from biological macromolecules and their complexes in solution. In: Comprehensive biophysics. Elsevier, Amsterdam, pp 376–397. CrossRefGoogle Scholar
  10. Ellis J, Gutierrez A, Barsukov IL, Huang WC, Grossmann JG, Roberts GC (2009) Domain motion in cytochrome P450 reductase: conformational equilibria revealed by NMR and small-angle x-ray scattering. J Biol Chem 284(52):36628–36637. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Fischer H, De Oliveira NM, Napolitano HB, Polikarpov I, Craievich A (2010) Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale. J Appl Crystallogr 43:101–109. CrossRefGoogle Scholar
  12. Forster F, Webb B, Krukenberg KA, Tsuruta H, Agard DA, Sali A (2008) Integration of small-angle X-ray scattering data into structural modeling of proteins and their assemblies. J Mol Biol 382(4):1089–1106. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gillespie JR, Shortle D (1997) Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. Paramagnetic relaxation enhancement by nitroxide spin labels. J Mol Biol 268(1):158–169. CrossRefPubMedGoogle Scholar
  14. Glatter O (1977) A new method for the evaluation of small-angle scattering data. J Appl Crystallogr 10(5):415–421. CrossRefGoogle Scholar
  15. Glatter O, Kratky O (1982) Small angle X-ray scattering in. Academic, New York, p 515Google Scholar
  16. Graewert MA, Svergun DI (2013) Impact and progress in small and wide angle X-ray scattering (SAXS and WAXS). Curr Opin Struct Biol 23(5):748–754. 10.1016/ CrossRefPubMedGoogle Scholar
  17. Grant TD, Luft JR, Wolfley JR, Tsuruta H, Martel A, Montelione GT, Snell EH (2011) Small angle X-ray scattering as a complementary tool for high-throughput structural studies. Biopolymers 95(8):517–530. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 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):16621–16628. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Guinier A, Fournet G (1955) Small-angle scattering of X-rays. WileyGoogle Scholar
  20. Hammel M (2012) Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS). Eur Biophys J 41(10):789–799. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hennig J, Sattler M (2014) The dynamic duo: combining NMR and small angle scattering in structural biology. Protein Sci 23(6):669–682. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature 450(7172):964–972. CrossRefPubMedGoogle Scholar
  23. Hubbell WL, Cafiso DS, Altenbach C (2000) Identifying conformational changes with site-directed spin labeling. Nat Struct Biol 7(9):735–739. CrossRefPubMedGoogle Scholar
  24. Hura GL, Menon AL, Hammel M, Rambo RP, Poole FL 2nd, Tsutakawa SE, Jenney FE Jr, Classen S, Frankel KA, Hopkins RC, Yang SJ, Scott JW, Dillard BD, Adams MW, Tainer JA (2009) Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS). Nat Methods 6(8):606–612. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jeffries CM, Trewhella J (2013) Small-angle scattering. In: Wall ME (ed) Quantitative biology: from molecular to celular systems. CRC Press, Boca Raton, FLGoogle Scholar
  26. Karplus M (1963) Vicinal proton coupling in nuclear magnetic resonance. J Am Chem Soc 85:2870–2871CrossRefGoogle Scholar
  27. Kikhney AG, Svergun DI (2015) A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett 589(19 Pt A):2570–2577. CrossRefGoogle Scholar
  28. 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
  29. Lee D, Walsh JD, Yu P, Markus MA, Choli-Papadopoulou T, Schwieters CD, Krueger S, Draper DE, Wang YX (2007) The structure of free L11 and functional dynamics of L11 in free, L11-rRNA(58 nt) binary and L11-rRNA(58 nt)-thiostrepton ternary complexes. J Mol Biol 367(4):1007–1022. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lemak A, Wu B, Yee A, Houliston S, Lee HW, Gutmanas A, Fang X, Garcia M, Semesi A, Wang YX, Prestegard JH, Arrowsmith CH (2014) Structural characterization of a flexible two-domain protein in solution using small angle X-ray scattering and NMR data. Structure 22(12):1862–1874. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lipfert J, Doniach S (2007) Small-angle X-ray scattering from RNA, proteins, and protein complexes. Annu Rev Biophys Biomol Struct 36:307–327. annurev.biophys.36.040306.132655 CrossRefPubMedGoogle Scholar
  32. Martel A, Liu P, Weiss TM, Niebuhr M, Tsuruta H (2012) An integrated high-throughput data acquisition system for biological solution X-ray scattering studies. J Synchrotron Radiat 19(Pt 3):431–434. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Mehmood S, Allison TM, Robinson CV (2015) Mass spectrometry of protein complexes: from origins to applications. Annu Rev Phys Chem 66:453–474. physchem-040214-121732 CrossRefPubMedGoogle Scholar
  34. Mylonas E, Svergun DI (2007) Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering. J Appl Crystallogr 40 (s1):s245–s249. 10.1107/S002188980700252X CrossRefGoogle Scholar
  35. Pelikan M, Hura GL, Hammel M (2009) Structure and flexibility within proteins as identified through small angle X-ray scattering. Gen Physiol Biophys 28(2):174–189CrossRefGoogle Scholar
  36. Putnam CD, Hammel M, Hura GL, Tainer JA (2007) X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys 40(3):191–285. CrossRefPubMedGoogle Scholar
  37. Rambo R BIOISIS ScÅtter. Accessed December 15, 2017
  38. Rambo RP, Tainer JA (2011) Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95(8):559–571. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Rozycki B, Kim YC, Hummer G (2011) SAXS ensemble refinement of ESCRT-III CHMP3 conformational transitions. Structure 19(1):109–116. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Schneidman-Duhovny D, Rossi A, Avila-Sakar A, Kim SJ, Velazquez-Muriel J, Strop P, Liang H, Krukenberg KA, Liao M, Kim HM, Sobhanifar S, Dotsch V, Rajpal A, Pons J, Agard DA, Cheng Y, Sali A (2012) A method for integrative structure determination of protein-protein complexes. Bioinformatics 28(24):3282–3289. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 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(5):1152–1166. CrossRefPubMedGoogle Scholar
  42. Sekhar A, Kay LE (2013) NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. Proc Natl Acad Sci U S A 110(32):12867–12874. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Svergun DI, Koch MHJ (2003) Small-angle scattering studies of biological macromolecules in solution. Rep Prog Phys 66(10):1735–1782CrossRefGoogle Scholar
  44. Thompson MK, Ehlinger AC, Chazin WJ (2017) Analysis of functional dynamics of modular multidomain proteins by SAXS and NMR. Methods Enzymol 592:49–76. CrossRefPubMedGoogle Scholar
  45. Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278(5340):1111–1114CrossRefGoogle Scholar
  46. 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(30):10507–10515CrossRefGoogle Scholar
  47. Yang S, Blachowicz L, Makowski L, Roux B (2010) Multidomain assembled states of Hck tyrosine kinase in solution. Proc Natl Acad Sci U S A 107(36):15757–15762. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Department of Structural BiologyUniversity of Pittsburgh School of MedicinePittsburghUSA

Personalised recommendations