Dengue pp 241-252 | Cite as

Use of Small-Angle X-ray Scattering to Investigate the Structure and Function of Dengue Virus NS3 and NS5

  • Kyung H. ChoiEmail author
  • Marc Morais
Part of the Methods in Molecular Biology book series (MIMB, volume 1138)


Small-angle X-ray scattering (SAXS) is a powerful reemerging biophysical technique that can be used to directly analyze many properties related to the size and shape of a macromolecule in solution. For example, the radius of gyration and maximum diameter of a macromolecule can be readily extracted from SAXS data, as can information regarding how well folded a protein is. Similarly, the molecular weight of macromolecular complexes can be directly determined from the complex’s scattering profile, providing insight into the oligomeric state and stoichiometry of the assembly. Furthermore, recently developed procedures for ab initio shape determination can provide low-resolution (~20 Å) molecular envelopes of proteins/complexes in their native state. In conjunction with high-resolution structural data, more sophisticated analysis of SAXS data can help address questions regarding conformational change, molecular flexibility, and populations of states within molecular ensembles. Because SAXS samples are easy to prepare and SAXS data is relatively easy to collect, the technique holds great promise for investigating the structure of macromolecules and their assemblies as well as monitoring and modeling their conformational changes. Here we describe typical steps in SAXS sample preparation and data collection and analysis and provide examples of SAXS analysis to investigate the structure and function of dengue virus NS3 and NS5.

Key words

Small-angle X-ray scattering Ab initio shape determination Radius of gyration Pair-wise distribution function 



This work is supported by NIH grant AI087856 to KHC and GM095516 to MCM. We thank Dr. Mark White and Dr. Cecile Bussetta for providing SAXS examples [19] and for helpful discussions.


  1. 1.
    Guinier A (1939) La diffraction des rayons X aux tres petits angles; application a l’etude de phenomenes ultramicroscopiques. Ann Phys (Paris) 12:161–237Google Scholar
  2. 2.
    Svergun DI, Petoukhov MV, Koch MHJ (2001) Determination of domain structure of proteins from X-ray solution scattering. Biophys J 80(6):2946–2953PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr 36:1277–1282CrossRefGoogle Scholar
  4. 4.
    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):191–285PubMedCrossRefGoogle Scholar
  5. 5.
    Bussetta C, Choi KH (2012) Dengue virus nonstructural protein 5 adopts multiple conformations in solution. Biochemistry 51(30):5921–5931PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Mertens HD, Svergun DI (2010) Structural characterization of proteins and complexes using small-angle X-ray solution scattering. J Struct Biol 172(1):128–141PubMedCrossRefGoogle Scholar
  7. 7.
    Mylonas E, Svergun DI (2007) Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering. J Appl Crystallogr 40:S245–S249CrossRefGoogle Scholar
  8. 8.
    Svergun DI (1992) Determination of the Regularization Parameter in Indirect-Transform Methods Using Perceptual Criteria. J Appl Crystallogr 25:495–503CrossRefGoogle Scholar
  9. 9.
    Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing (vol 76, p 2879, 1999). Biophys J 77(5):2896–2896CrossRefGoogle Scholar
  10. 10.
    Volkov VV, Svergun DI (2003) Uniqueness of ab initio shape determination in small-angle scattering. J Appl Crystallogr 36:860–864CrossRefGoogle Scholar
  11. 11.
    Kozin MB, Svergun DI (2001) Automated matching of high- and low-resolution structural models. J Appl Crystallogr 34:33–41CrossRefGoogle Scholar
  12. 12.
    Wriggers W (2010) Using Situs for the integration of multi-resolution structures. Biophys Rev 2(1):21–27PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Mastrangelo E, Milani M, Bollati M, Selisko B, Peyrane F, Pandini V, Sorrentino G, Canard B, Konarev PV, Svergun DI, de Lamballerie X, Coutard B, Khromykh AA, Bolognesi M (2007) Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein. J Mol Biol 372(2):444–455PubMedCrossRefGoogle Scholar
  14. 14.
    Petoukhov MV, Svergun DI (2005) Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys J 89(2):1237–1250PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Svergun D, Barberato C, Koch MHJ (1995) CRYSOL—a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773CrossRefGoogle Scholar
  16. 16.
    Luo D, Xu T, Hunke C, Gruber G, Vasudevan SG, Lescar J (2008) Crystal structure of the NS3 protease-helicase from dengue virus. J Virol 82(1):173–183PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, Owens RJ, Stuart DI, Grimes JM, Mancini EJ (2009) Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol 83(24):12895–12906PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    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):5656–5664PubMedCrossRefGoogle Scholar
  19. 19.
    Zheng J, Gay DC, Demeler B, White MA, Keatinge-Clay AT (2012) Divergence of multimodular polyketide synthases revealed by a didomain structure. Nat Chem Biol 8(7):615–621PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Texas Medical BranchGalvestonUSA

Personalised recommendations