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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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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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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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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DOI: https://doi.org/10.1038/nprot.2016.113
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