Sample Preparation, Data Collection, and Preliminary Data Analysis in Biomolecular Solution X‐Ray Scattering

Alexander Grishaev1

1 Laboratory of Chemical Physics, National Institutes of Health, NIDDK, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.14
DOI:  10.1002/0471140864.ps1714s70
Online Posting Date:  November, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


In addition to the classic methods of structural biology—X‐ray crystallography and NMR—solution X‐ray scattering (SAXS) is playing an increasingly important role in structural investigation of biological macromolecules. However, the simultaneous ease of SAXS data collection and sophistication of its data analysis tools can present challenges to the investigator. Any sample, whether pure or contaminated, whether monodisperse or polydisperse, will yield scattering data, and it is up to the user to ensure the absence of artifacts and to choose a proper structural modeling strategy. This unit discusses experimental aspects of X‐ray solution scattering, including sample preparation and data collection, as well as the steps in data processing and preliminary analysis required to ensure the absence of artifacts. The goal is to summarize everything than can go wrong with SAXS data measurement so the user can have confidence in the data before undertaking structural modeling. Curr. Protoc. Protein Sci. 70:17.14.1‐17.14.18. © 2012 by John Wiley & Sons, Inc.

Keywords: solution X‐ray scattering; SAXS; WAXS; sample preparation; aggregation; radiation damage; protein and RNA structure; structural biology

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Preparation of the Matching Buffer for SAXS Measurements
  • Buffer Composition Guidelines for SAXS Samples
  • Final Steps in SAXS Sample Preparation
  • SAXS Data Measurement: Laboratory‐Based Instruments Versus Synchrotron Beam Lines
  • SAXS Data Measurement on a Laboratory‐Based Instrument
  • Synchrotron SAXS Data Measurement: q‐Range Selection(s)
  • Measurements of the Synchrotron SAXS Data: Preliminary Steps
  • Summary
  • Acknowledgements
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Bernado, P., Mylonas, E., Petoukhov, M.V., Blackledge, M., and Svergun, D.I. 2007. Structural characterization of flexible proteins using small‐angle x‐ray scattering. J. Am. Chem. Soc. 129:5656‐5664.
   Chacon, P., Moran, F., Diaz, J.F., Pantos, E., and Andreu, J.M. 1998. Low‐resolution structures of proteins in solution retrieved from x‐ray scattering with a genetic algorithm. Biophys. J. 74:2760‐2775.
   Fritz, G., Bergmann, A., and Glatter, O. 2000. Evaluation of small‐angle scattering data of charged particles using the generalized indirect Fourier transformation technique. J. Chem. Phys. 113:9733‐9740.
   Grishaev, A., Wu, J., Trewhella, J., and Bax, A. 2005. Refinement of multidomain protein structures by combination of solution small‐angle x‐ray scattering and NMR data. J. Am. Chem. Soc. 127:16621‐16628.
   Grishaev, A., Tugarinov, V., Kay, L.E., Trewhella, J., and Bax, A. 2008a. Refined solution structure of the 82‐kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints. J. Biomol. NMR 40:95‐106.
   Grishaev, A., Ying, J., Canny, M., Pardi, A., and Bax, A. 2008b. Solution structure of tRNA(Val) from refinement of homology model against residual dipolar couplings and SAXS data. J. Biomol. NMR 42:99‐109.
   Grishaev, A., Guo, L., Irving, T., and Bax, A. 2010. Improved fitting of solution x‐ray scattering data to macromolecular structural ensembles by explicit water modeling. J. Am. Chem. Soc. 132:15484‐15486.
   Jacques, D.A. and Trewhella, J. 2010. Small‐angle scattering for structural biology expanding the frontier while avoiding the pitfalls. Prot. Sci. 19:642‐657.
   Koch, M.H.J., Vachette, P., and Svergun, D.I. 2003. Small‐angle scattering: A view on the properties, structures and structural changes of biological macromolecules in solution. Q. Rev. Biophys. 36:147‐227.
   Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H.J., and Svergun, D.I. 2003. PRIMUS: a Windows‐PC based system for small‐angle scattering data analysis. J. Appl. Crystallogr. 36:1277‐1282.
   Mattinen, M.L., Paakkonen, K., Ikonen, T., Craven, J., Drakenberg, T., Serimaa, R., Waltho, J., and Annilla, A. 2003. Quaternary structure built from subunits combining NMR and small‐angle x‐ray scattering data. Biophys. J. 83,1177‐1183.
   Mittag, T., Marsh, J., Grishaev, A., Orlicky, S., Lin, H., Sicheri, F., Tyers, M., and Forman‐Kay, J. 2010. Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase. Structure 18:494‐506.
   Mylonas, E. and Svergun, D.I. 2007. Accuracy of molecular mass determination of proteins in solution by small‐angle X‐ray scattering. J. Appl. Crystallogr. 40:s245‐s249.
   Orthaber, D., Bergmann, A., and Glatter, O. 2000. SAXS experiments on absolute scale with Kratky systems using water as a secondary standard. J. Appl. Crystallogr. 33:218‐225.
   Pelikan, M., Hura, G.L., Hammel, M. 2009. Structure and flexibility within proteins identified through small‐angle X‐ray scattering. Gen. Physiol. Biophys. 28:174‐189.
   Petoukhov, M.V. and Svergun, D.I. 2005. Global rigid body modeling of macromolecular complexes against small‐angle scattering data. Biophys. J. 89,1237‐1250.
   Petoukhov, M.V. and Svergun, D.I. 2007. Analysis of X‐ray and neutron scattering from biomacromolecular solutions. Curr. Opin. Struct. Biol. 17:562‐571.
   Poitevin, F., Orland, H., Doniach, S., Koehl, P., and Delarue, M. 2011. AquaSAXS: A web server for computation and fitting of SAXS profiles with non‐uniformly hydrated atomic models. Nucl. Acids Res. 39:W184‐W189.
   Putnam, C.D., Hammel, M., Hura, G., and Tainer, J.A. 2007. X‐ray solution scattering (SAXS) combined with crystallography and computation: Defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys. 40:191‐285.
   Schneiderman‐Duhovny, D., Hammel, M., and Sali, A. 2010. FoXS: A web server for rapid computation and fitting of SAXS profiles. Nucl. Acids Res. 28:W540‐W544.
   Schwieters, C.D. and Clore, G.M. 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 46:1152‐1166.
   Schwieters, C.D., Suh, J‐Y., Grishaev, A., Ghirlando, R., Takayama, Y., and Clore, G.M. 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. 132:13026‐13045.
   Svergun, D.I. 1992. Determination of the regularization parameter in indirect‐transform methods using perceptual criteria. J. Appl. Crystallogr. 25:495‐503.
   Svergun, D., Barberato, C., and Koch, M.H.J. 1995. CRYSOL a program to evaluate x‐ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28:768‐773.
   Svergun, D.I. 1999. Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76:2879‐2886.
   Svergun, D.I., Petoukhov, M.V., and Koch, M.H.J. 2001. Determination of domain structure of proteins from x‐ray solution scattering. Biophys. J. 80:2001.
   Takahashi, Y., Nishikawa, Y., and Fujisawa, T. 2003. Evaluation of three algorithms for ab initio determination of three‐dimensional shape from one‐dimensional solution scattering profiles. J. Appl. Crystallogr. 36:549‐552.
   Takayama, Y., Schwieters, C.D., Grishaev, A., Ghirlando, R., and Clore, G.M. 2011. Combined use of residual dipolar couplings and solution x‐ray scattering to rapidly probe rigid‐body conformational transitions in a non‐phosphorylatable active‐site Mutant of the 128 kDa Enzyme I dimer. J. Am. Chem. Soc. 133:424‐427.
   Volkov, V.V. and Svergun, D.I. 2003. Uniqueness of ab initio shape determination in small‐angle scattering. J. Appl. Crystallogr. 36:860‐864.
   Walther, D., Cohen, F.E., and Doniach, S. 2000. Reconstruction of low‐resolution three‐dimensional density maps from one‐dimensional small‐angle X‐ray scattering data for biomolecules. J. Appl. Crystallogr. 33:350‐363.
   Yang, S., Park, S., Makowski, L., and Roux, B. 2009. A rapid coarse residue‐based computational method for X‐ray solution scattering characterization of protein folds and multiple conformational states of large protein complexes. Biophys. J. 96:4449‐4463.
PDF or HTML at Wiley Online Library