Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Synchrotron-based small-angle X-ray scattering of proteins in solution

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

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

Figure 1: A basic SAXS setup.
Figure 2: Size-exclusion chromatogram of HSA.
Figure 3: Typical scattering from protein solution (red) and the corresponding buffer (gray).
Figure 4: Examples of good and bad buffer subtractions.
Figure 5: Comparison of HSA with and without the final purification step.
Figure 6: Example of Guinier analysis.
Figure 7: Concentration effects on Rg and I(0).
Figure 8: The pair distance distribution function, P(r), is sensitive to concentration effects and sample polydispersity.
Figure 9: Comparisons to a crystal structure.

Similar content being viewed by others

References

  1. Nagar, B. & Kuriyan, J. SAXS and the working protein. Structure 13, 169–170 (2005).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. Ando, N. et al. Transient B12-dependent methyltransferase complexes revealed by small-angle X-ray scattering. J. Am. Chem. Soc. 134, 17945–17954 (2012).

    Article  CAS  Google Scholar 

  4. Ando, N. et al. Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 108, 21046–21051 (2011).

    Article  CAS  Google Scholar 

  5. 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).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. Jacques, D.A. & Trewhella, J. Small-angle scattering for structural biology: expanding the frontier while avoiding the pitfalls. Protein Sci. 19, 642–657 (2010).

    Article  CAS  Google Scholar 

  9. 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).

    Article  CAS  Google Scholar 

  10. Pollack, L. SAXS Studies of ion-nucleic acid interactions. Annu. Rev. Biophys. 40, 225–242 (2011).

    Article  CAS  Google Scholar 

  11. 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).

    Article  CAS  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. 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).

    Article  Google Scholar 

  14. Glatter, O. & Kratky, O. Small Angle X-ray Scattering (Academic Press, 1982).

  15. Svergun, D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992).

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Hura, G.L. et al. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS). Nat. Methods 6, 606–612 (2009).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Mathew, E., Mirza, A. & Menhart, N. Liquid-chromatography-coupled SAXS for accurate sizing of aggregating proteins. J. Synchrotr. Radiat. 11, 314–318 (2004).

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. Pernot, P. et al. New beamline dedicated to solution scattering from biological macromolecules at the ESRF. J. Phys. Confer. Ser. 247, 012009 (2010).

    Article  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Akiyama, S. Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering. J. Appl. Crystallogr. 43, 237–243 (2010).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. Kozak, M. Glucose isomerase from Streptomyces rubiginosus: potential molecular weight standard for small-angle X-ray scattering. J. Appl. Cryst. 38, 555–558 (2005).

    Article  CAS  Google Scholar 

  30. Murphy, B.M. et al. Protein instability following transport or storage on dry ice. Nat. Methods 10, 278–279 (2013).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. Moore, P.B. Small-angle scattering: information content and error analysis. J. Appl. Crystallogr. 13, 168–175 (1980).

    Article  CAS  Google Scholar 

  33. Gherardi, E. et al. Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proc. Natl. Acad. Sci. USA 103, 4046–4051 (2006).

    Article  CAS  Google Scholar 

  34. Svergun, D.I. Restoring low-resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76, 2879–2886 (1999).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. 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).

    Article  Google Scholar 

  37. Rambo, R.P. & Tainer, J.A. Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496, 477–481 (2013).

    Article  CAS  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

  40. 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).

    Article  CAS  Google Scholar 

  41. 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).

    Article  CAS  Google Scholar 

  42. Curry, S. Lessons from the crystallographic analysis of small molecule binding to human serum albumin. Drug Metab. Pharmacokinetics 24, 342–357 (2009).

    Article  CAS  Google Scholar 

  43. 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).

    Article  CAS  Google Scholar 

  44. Volkov, V.V. & Svergun, D.I. Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864 (2003).

    Article  CAS  Google Scholar 

  45. 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).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. Chen, H. et al. Ionic strength-dependent persistence lengths of single-stranded RNA and DNA. Proc. Natl. Acad. Sci. USA 109, 799–804 (2012).

    Article  CAS  Google Scholar 

Download references

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

Authors

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

Correspondence to Nozomi Ando.

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

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.116

This article is cited by

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing