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Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments

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Abstract

Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume, including the solvent and buffer components, as well as the macromolecules of interest. To obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis, so it is essential that the samples be pure and monodisperse for the duration of the experiment. This protocol outlines the basic physics of SAXS and SANS, and it reveals how the underlying conceptual principles of the techniques ultimately 'translate' into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size-exclusion chromatography (SEC) and light scattering. Also included are procedures that are specific to X-rays (in-line SEC–SAXS) and neutrons, specifically preparing samples for contrast matching or variation experiments and deuterium labeling of proteins.

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Figure 1: Scattering basics.
Figure 2: Decreasing contrast (Δρ) and the effect on measured scattering intensities.
Figure 3: Coherent and incoherent neutron scattering.
Figure 4: Principle of contrast matching.
Figure 5: The effect of nonexchangeable deuterium labeling of a component for SANS with contrast-variation experiments.
Figure 6: Sample purity and contaminants (simulated SAXS data and simulated SDS–PAGE).
Figure 7: The value of native PAGE.
Figure 8: Sample characterization: SDS–PAGE combined with SEC.
Figure 9: DLS as a tool for characterizing samples and sample handling.
Figure 10: The importance of obtaining matched sample solvents.
Figure 11: MULCh calculations of component X-ray and neutron contrasts.
Figure 12: Dialysis setup for SANS.
Figure 13: Troubleshooting.
Figure 14: SEC–SAXS component separation.

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Acknowledgements

This work was supported by the Bundesministerium für Bildung und Forschung (BMBF) project BIOSCAT, grant 05K12YE1, by the European Community' Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement no. 283570) and by an HFSP grant (RGP0017/2012 to D.I.S. and C.M.J.). M.A.G. was supported by an EMBL Interdisciplinary Postdoc Programme (EIPOD) and Marie Curie COFUND actions. We thank J. Trewhella, in whose laboratory many of the procedures were performed. We also thank A. Duff (Australian Nuclear Science and Technology Organisation (ANSTO)) and David Jacques (LMB, Cambridge) for constructive comments on the deuteration incorporation spreadsheet.

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Contributions

C.M.J., M.A.G., C.E.B., D.B.L., A.E.W. and D.I.S. helped develop SAXS and SANS sample preparation protocols and analytical tools. C.M.J., M.A.G., C.E.B. and D.I.S. performed radiation damage studies and developed protocols for SEC–SAXS. C.M.J., A.E.W. and D.B.L. contributed to 'in-house' 2H-labeling protocols. D.B.L., A.E.W., C.M.J. and D.I.S. optimized protocols for preparing samples for SANS with contrast variation. A.E.W. developed Contrast. C.M.J., M.A.G., C.E.B., D.B.L., A.E.W. and D.I.S. critically discussed and wrote the manuscript.

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Correspondence to Cy M Jeffries.

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The authors declare no competing financial interests. Although particular commercial products are noted throughout the text, such references should not be interpreted as product endorsements.

Supplementary information

Supplementary Method 1

Calculating molecular weight from absolute scaled data (XLSX 178 kb)

Supplementary Method 2

Calculating 2H labeling of a protein (XLSX 18 kb)

Supplementary Data

Contrast module input file (TXT 0 kb)

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Jeffries, C., Graewert, M., Blanchet, C. et al. Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments. Nat Protoc 11, 2122–2153 (2016). https://doi.org/10.1038/nprot.2016.113

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