Abstract
With recent advances in data analysis algorithms, X-ray detectors and synchrotron sources, small-angle X-ray scattering (SAXS) has become much more accessible to the structural biology community. Although limited to ∼10 Å resolution, SAXS can provide a wealth of structural information on biomolecules in solution and is compatible with a wide range of experimental conditions. SAXS is thus an attractive alternative when crystallography is not possible. Moreover, advanced use of SAXS can provide unique insight into biomolecular behavior that can only be observed in solution, such as large conformational changes and transient protein-protein interactions. Unlike crystal diffraction data, however, solution scattering data are subtle in appearance, highly sensitive to sample quality and experimental errors and easily misinterpreted. In addition, synchrotron beamlines that are dedicated to SAXS are often unfamiliar to the nonspecialist. Here we present a series of procedures that can be used for SAXS data collection and basic cross-checks designed to detect and avoid aggregation, concentration effects, radiation damage, buffer mismatch and other common problems. Human serum albumin (HSA) serves as a convenient and easily replicated example of just how subtle these problems can sometimes be, but also of how proper technique can yield pristine data even in problematic cases. Because typical data collection times at a synchrotron are only one to several days, we recommend that the sample purity, homogeneity and solubility be extensively optimized before the experiment.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Nagar, B. & Kuriyan, J. SAXS and the working protein. Structure 13, 169–170 (2005).
Putnam, C.D., Hammel, M., Hura, G.L. & Tainer, J.A. 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 (2007).
Ando, N. et al. Transient B12-dependent methyltransferase complexes revealed by small-angle X-ray scattering. J. Am. Chem. Soc. 134, 17945–17954 (2012).
Ando, N. et al. Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 108, 21046–21051 (2011).
Rambo, R.P. & Tainer, J.A. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95, 559–571 (2011).
Pollack, L. et al. Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle X-ray scattering. Proc. Natl. Acad. Sci. USA 96, 10115–10117 (1999).
Pollack, L. et al. Time resolved collapse of a folding protein observed with small angle X-ray scattering. Phys. Rev. Lett. 86, 4962–4965 (2001).
Jacques, D.A. & Trewhella, J. Small-angle scattering for structural biology: expanding the frontier while avoiding the pitfalls. Protein Sci. 19, 642–657 (2010).
Nielsen, S.S. et al. BioXTAS RAW, a software program for high-throughput automated small-angle X-ray scattering data reduction and preliminary analysis. J. Appl. Crystallogr. 42, 959–964 (2009).
Pollack, L. SAXS Studies of ion-nucleic acid interactions. Annu. Rev. Biophys. 40, 225–242 (2011).
Graewert, M.A. & Svergun, D.I. Impact and progress in small and wide angle X-ray scattering (SAXS and WAXS). Curr. Opin. Struct. Biol. 23, 748–754 (2013).
Jacques, D.A., Guss, J.M., Svergun, D.I. & Trewhella, J. Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution. Acta Crystallogr. Sect. D 68, 620–626 (2012).
Pérez, J. & Nishino, Y. Advances in X-ray scattering: from solution SAXS to achievements with coherent beams. Curr. Opin. Struct. Biol. 22, 670–678 (2012).
Glatter, O. & Kratky, O. Small Angle X-ray Scattering (Academic Press, 1982).
Svergun, D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992).
Konarev, P.V., Petoukhov, M.V., Volkov, V.V. & Svergun, D.I. ATSAS 2.1, a program package for small-angle scattering data analysis. J. Appl. Crystallogr. 39, 277–286 (2006).
Konarev, P.V., Volkov, V.V., Sokolova, A., Koch, M.H.J. & Svergun, D.I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003).
Schneidman-Duhovny, D., Hammel, M. & Sali, A. FoXS: A web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 38, W540–W544 (2010).
Hura, G.L. et al. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS). Nat. Methods 6, 606–612 (2009).
Lafleur, J.P. et al. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering. J. Appl. Crystallogr. 44, 1090–1099 (2011).
Mathew, E., Mirza, A. & Menhart, N. Liquid-chromatography-coupled SAXS for accurate sizing of aggregating proteins. J. Synchrotr. Radiat. 11, 314–318 (2004).
Nielsen, S.S., Møller, M. & Gillilan, R.E. High-throughput biological small-angle X-ray scattering with a robotically loaded capillary cell. J. Appl. Crystallogr. 45, 213–223 (2012).
Pernot, P. et al. New beamline dedicated to solution scattering from biological macromolecules at the ESRF. J. Phys. Confer. Ser. 247, 012009 (2010).
Round, A.R. et al. Automated sample-changing robot for solution scattering experiments at the EMBL Hamburg SAXS station X33. J. Appl. Crystallogr. 41, 913–917 (2008).
Fischetti, R.F. et al. High-resolution wide-angle X-ray scattering of protein solutions: effect of beam dose on protein integrity. J. Synchrotr. Radiat. 10, 398–404 (2003).
Kuwamoto, S., Akiyama, S. & Fujisawa, T. Radiation damage to a protein solution, detected by synchrotron X-ray small-angle scattering: dose-related considerations and suppression by cryoprotectants. J. Synchrotr. Radiat. 11, 462–468 (2004).
Akiyama, S. Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering. J. Appl. Crystallogr. 43, 237–243 (2010).
Mylonas, E. & Svergun, D.I. Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering. J. Appl. Crystallogr. 40, s245–s249 (2007).
Kozak, M. Glucose isomerase from Streptomyces rubiginosus: potential molecular weight standard for small-angle X-ray scattering. J. Appl. Cryst. 38, 555–558 (2005).
Murphy, B.M. et al. Protein instability following transport or storage on dry ice. Nat. Methods 10, 278–279 (2013).
Orthaber, D., Bergmann, A. & Glatter, O. SAXS experiments on absolute scale with Kratky systems using water as a secondary standard. J. Appl. Crystallogr. 33, 218–225 (2000).
Moore, P.B. Small-angle scattering: information content and error analysis. J. Appl. Crystallogr. 13, 168–175 (1980).
Gherardi, E. et al. Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proc. Natl. Acad. Sci. USA 103, 4046–4051 (2006).
Svergun, D.I. Restoring low-resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76, 2879–2886 (1999).
Franke, D. & Svergun, D.I. DAMMIF, a program for rapid ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342–346 (2009).
Fischer, H., Oliveira Neto, M.d., Napolitano, H.B., Polikarpov, I. & Craievich, A.F. 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 (2009).
Rambo, R.P. & Tainer, J.A. Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496, 477–481 (2013).
Petoukhov, M.V. et al. New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342–350 (2012).
Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423 (1995).
Tate, M.W. et al. A large-format high-resolution area X-ray detector based on a fiber-optically bonded charge-coupled device (CCD). J. Appl. Crystallogr. 28, 196–205 (1995).
Ando, N., Chenevier, P., Novak, M., Tate, M.W. & Gruner, S.M. High hydrostatic pressure small-angle X-ray scattering cell for protein solution studies featuring diamond windows and disposable sample cells. J. Appl. Crystallogr. 41, 167–175 (2008).
Curry, S. Lessons from the crystallographic analysis of small molecule binding to human serum albumin. Drug Metab. Pharmacokinetics 24, 342–357 (2009).
Koch, M.H.J., Vachette, P. & Svergun, D.I. Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution. Q. Rev. Biophys. 36, 147–227 (2003).
Volkov, V.V. & Svergun, D.I. Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864 (2003).
Bhattacharya, A.A., Grüne, T. & Curry, S. Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin. J. Mol. Biol. 303, 721–732 (2000).
Svergun, D.I., Barberato, C. & Koch, M.H.J. CRYSOL: a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).
Chen, H. et al. Ionic strength-dependent persistence lengths of single-stranded RNA and DNA. Proc. Natl. Acad. Sci. USA 109, 799–804 (2012).
Acknowledgements
We thank S. Meisburger (Cornell University) for providing the MATLAB code that was used for integration of representative data. This work is based upon research conducted at the G1 Station of the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation (NSF) and the National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) under NSF award DMR-0936384, using the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by NIH/NIGMS award GM-103485. This work was supported by NIH grant K99GM100008 to N.A.
Author information
Authors and Affiliations
Contributions
N.A. designed the experiment. N.A. and R.E.G. collected data. S.S. and N.A. performed data analysis. N.A., S.S. and R.E.G. wrote the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Planning a concentration series experiment.
To perform a concentration series experiment, proteins solutions must be prepared at three or more protein concentrations in the same buffer (here, represented as protein solutions #1-3). For each sample, the total volume should be equal to or greater than the minimum volume required by the sample cell. Recipes should be designed such that mixing volumes can be pipetted accurately. Multiple X-ray exposures are collected on each protein solution and matching buffer as described in the Procedures. Data are then reduced (i.e. by averaging and subtraction) in order to produce a single background-subtracted scattering profile, from which structural information can be gained. Multiple background-subtracted scattering profiles at different protein concentrations are then required to examine concentration effects and estimate structural parameters, such as Rg at infinite dilution. As many exposures are generated in a single experiment, careful and detailed documentation is indispensable.
Supplementary information
Supplementary Figure 1
Planning a concentration series experiment. (PDF 124 kb)
Supplementary Data: Example data on HSA.
The compressed archive file contains all buffer-subtracted profiles introduced in the Anticipated Results section as well as examples of unsubtracted, unaveraged profiles. Data files are in ASCII format with columns corresponding to q, I, and the standard deviation of I. (ZIP 560 kb)
Representative shape reconstruction
The simulated annealing of a typical HSA bead model in DAMMIF35 is shown. Note that although a crystal structure is superimposed for comparison, individual reconstructions prior to averaging should not be over-interpreted. (MOV 12612 kb)
Rights and permissions
About this article
Cite this article
Skou, S., Gillilan, R. & Ando, N. Synchrotron-based small-angle X-ray scattering of proteins in solution. Nat Protoc 9, 1727–1739 (2014). https://doi.org/10.1038/nprot.2014.116
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2014.116
This article is cited by
-
Subdomain dynamics enable chemical chain reactions in non-ribosomal peptide synthetases
Nature Chemistry (2024)
-
Scrutinizing the protein hydration shell from molecular dynamics simulations against consensus small-angle scattering data
Communications Chemistry (2023)
-
Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV
Analytical and Bioanalytical Chemistry (2023)
-
Small-angle X-ray and neutron scattering
Nature Reviews Methods Primers (2021)
-
Protein intrinsic viscosity determination with the Viscosizer TD instrument: reaching beyond the initially expected applications
European Biophysics Journal (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.