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

Nature Protocols volume 11, pages 2122–2153 (2016)Cite this article

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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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References

  1. 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  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Skou, S., Gillilan, R.E. & Ando, N. Synchrotron-based small-angle X-ray scattering of proteins in solution. Nat. Protoc. 9, 1727–1739 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Petoukhov, M.V. et al. New developments in the ATSAS program package for small-angle scattering data analysis. J. App. Crystallogr. 45, 342–350 (2012).

    Article  CAS  Google Scholar 

  5. Petoukhov, M.V., Konarev, P.V., Kikhney, A.G. & Svergun, D.I. ATSAS 2.1 - towards automated and web-supported small-angle scattering data analysis. J. Appl. Crystallogr. 40, s223–s228 (2007).

    Article  CAS  Google Scholar 

  6. Franke, D., Kikhney, A.G. & Svergun, D.I. Automated acquisition and analysis of small angle X-ray scattering data. Nuc. Instr. Meth. Phys. Res. Sec. A 689, 52–59 (2012).

    Article  CAS  Google Scholar 

  7. Toft, K.N. et al. High-throughput small angle X-ray scattering from proteins in solution using a microfluidic front-end. Anal. Chem. 80, 3648–3654 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Blanchet, C.E. et al. Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY). J. Appl. Crystallogr. 48, 431–443 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Petoukhov, M.V. & Svergun, D.I. Applications of small-angle X-ray scattering to biomacromolecular solutions. Int. J. Biochem. Cell Biol. 45, 429–437 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Koch, M.H., 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  PubMed  Google Scholar 

  11. Wall, M.E., Gallagher, S.C. & Trewhella, J. Large-scale shape changes in proteins and macromolecular complexes. Ann. Rev. Phys. Chem. 51, 355–380 (2000).

    Article  CAS  Google Scholar 

  12. Mertens, H.D. & Svergun, D.I. Structural characterization of proteins and complexes using small-angle X-ray solution scattering. J. Struct. Biol. 172, 128–141 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Rambo, R.P. & Tainer, J.A. Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. Curr. Opin. Struct. Biol. 20, 128–137 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Krueger, S., Shin, J.H., Raghunandan, S., Curtis, J.E. & Kelman, Z. Atomistic ensemble modeling and small-angle neutron scattering of intrinsically disordered protein complexes: applied to minichromosome maintenance protein. Biophys. J. 101, 2999–3007 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Whitten, A.E. et al. The structure of the KinA-Sda complex suggests an allosteric mechanism of histidine kinase inhibition. J. Mol. Biol. 368, 407–420 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Vestergaard, B. et al. A helical structural nucleus is the primary elongating unit of insulin amyloid fibrils. PLoS Biol. 5, e134 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Valentini, E., Kikhney, A.G., Previtali, G., Jeffries, C.M. & Svergun, D.I. SASBDB, a repository for biological small-angle scattering data. Nucleic Acids Res. 43, D357–D363 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Svergun, D.I., Petoukhov, M.V. & Koch, M.H. Determination of domain structure of proteins from X-ray solution scattering. Biophys. J. 80, 2946–2953 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Petoukhov, M.V. & Svergun, D.I. Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys. J. 89, 1237–1250 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sali, A. et al. Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop. Structure 23, 1156–1167 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Panjkovich, A. & Svergun, D.I. Deciphering conformational transitions of proteins by small angle X-ray scattering and normal mode analysis. Phys. Chem. Chem. Phys. 18, 5707–5719 (2015).

    Article  CAS  Google Scholar 

  24. Hammel, M. Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS). Eur. Biophys. J. 41, 789–799 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bernado, P., Mylonas, E., Petoukhov, M.V., Blackledge, M. & Svergun, D.I. Structural characterization of flexible proteins using small-angle X-ray scattering. J. Am. Chem Soc. 129, 5656–5664 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, Y., Trewhella, J. & Goldenberg, D.P. Small-angle X-ray scattering of reduced ribonuclease A: effects of solution conditions and comparisons with a computational model of unfolded proteins. J. Mol. Biol. 377, 1576–1592 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Goldenberg, D.P. & Argyle, B. Minimal effects of macromolecular crowding on an intrinsically disordered protein: a small-angle neutron scattering study. Biophys. J. 106, 905–914 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tria, G., Mertens, H.D., Kachala, M. & Svergun, D.I. Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering. IUCrJ 2, 207–217 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kikhney, A.G. & Svergun, D.I. A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett. 589, 2570–2577 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Kachala, M., Valentini, E. & Svergun, D.I. Application of SAXS for the Structural Characterization of IDPs. Adv. Exp. Med. Biol. 870, 261–289 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Jimenez-Garcia, B., Pons, C., Svergun, D.I., Bernado, P. & Fernandez-Recio, J. pyDockSAXS: protein-protein complex structure by SAXS and computational docking. Nucleic Acids Res. 43, W356–W361 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cho, H.S., Schotte, F., Dashdorj, N., Kyndt, J. & Anfinrud, P.A. Probing anisotropic structure changes in proteins with picosecond time-resolved small-angle X-ray scattering. J. Phys. Chem. B 117, 15825–15832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Graceffa, R. et al. Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam. J. Synchrotron Radiat. 20, 820–825 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Roessle, M. et al. Time-resolved small angle scattering: kinetics and structural data from proteins in solution. J. Appl. Crystallogr. 33, 548–551 (2000).

    Article  CAS  Google Scholar 

  35. 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  PubMed  PubMed Central  Google Scholar 

  36. Grishaev, A. Sample preparation, data collection and preliminary data analysis in biomolecular solution X-ray scattering. Curr. Protoc. Protein Sci. 70, 17.14.1–17.14.18 (2012).

    Article  Google Scholar 

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

  38. Feigin, L.A. & Svergun, D.I. Structure Analysis by Small-angle X-ray and Neutron Scattering (Plenum Press, 1987).

  39. Svergun, D.I., Koch, M.H.J., Timmins, P.A. & May, R.P. Small Angle X-Ray and Neutron Scattering From Solutions of Biological Macromolecules. 1st edn, (Oxford University Press, 2013).

  40. 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. Sec. D68, 620–626 (2012).

    Article  CAS  Google Scholar 

  41. Svergun, D.I. & Nierhaus, K.H. A map of protein-rRNA distribution in the 70 S Escherichia coli ribosome. J. Biol. Chem. 275, 14432–14439 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Whitten, A.E., Jeffries, C.M., Harris, S.P. & Trewhella, J. Cardiac myosin-binding protein C decorates F-actin: implications for cardiac function. Proc. Natl. Acad. Sci. USA 105, 18360–18365 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Lakey, J.H. Neutrons for biologists: a beginner's guide, or why you should consider using neutrons. J. R. Soc. 6, S567–S573 (2009).

    CAS  Google Scholar 

  44. Zaccai, G. Straight lines of neutron scattering in biology: a review of basic controls in SANS and EINS. Eur. Biophys. J. 41, 781–787 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Krueger, S. SANS provides unique information on the structure and function of biological macromolecules in solution. Physica B 241-243, 1131–1137 (1997).

    Article  CAS  Google Scholar 

  46. Heller, W.T. & Littrell, K.C. Small-angle neutron scattering for molecular biology: basics and instrumentation. Meth. Mol. Biol. 544, 293–305 (2009).

    Article  CAS  Google Scholar 

  47. Krueger, J.K. & Wignall, G.D. Small-Angle Neutron Scattering from Biological Molecules. in Neutron Scattering in Biology 127–160 (Berlin/Heidelberg: Springer, 2006).

  48. Svergun, D.I. & Koch, M.H.J. Small-angle scattering studies of biological macromolecules in solution. Rep. Prog. Phys. 66, 1735–1782 (2003).

    Article  CAS  Google Scholar 

  49. Jacrot, B. & Zaccai, G. Determination of molecular weight by neutron scattering. Biopolymers 20, 2413–2426 (1981).

    Article  CAS  Google Scholar 

  50. Sears, V.F. Neutron scattering lengths and cross sections. Neutron News 3, 26–37 (1992).

    Article  Google Scholar 

  51. Whitten, A.E., Cai, S. & Trewhella, J. MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies. J. App. Crystallogr. 41, 222–226 (2008).

    Article  CAS  Google Scholar 

  52. Konarev, P.V. & Svergun, D.I. A posteriori determination of the useful data range for small-angle scattering experiments on dilute monodisperse systems. IUCrJ 2, 352–360 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Lafleur, J.P. et al. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering. J. App. Crystallogr. 44, 1090–1099 (2011).

    Article  CAS  Google Scholar 

  54. Skou, M., Skou, S., Jensen, T.G., Vestergaard, B. & Gillilan, R.E. icrofluidic dialysis for biological small-angle X-ray scattering. J. Appl. Crystallogr. 47, 1355–1366 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Bertani, G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293–300 (1951).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 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. Synchrotron Radiat. 11, 462–468 (2004).

    Article  CAS  PubMed  Google Scholar 

  57. Jeffries, C.M., Graewert, M.A., Svergun, D.I. & Blanchet, C.E. Limiting radiation damage for high brilliance biological solution scattering: practical experience at the EMBL P12 beamline, PETRAIII. J. Synchrotron Radiat. 22, 273–279 (2014).

    Article  CAS  Google Scholar 

  58. Folta-Stogniew, E. & Williams, K.R. Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J. Biomol. Tech. 10, 51–63 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Jacques, D.A. et al. Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state. Acta Crystallogr. D Biol. Crystallogr. 65, 574–581 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Dünweg, B., Reith, D., Steinhauser, M. & Kremer, K. Corrections to scaling in the hydrodynamic properties of dilute polymer solutions. J. Chem. Phys. 117, 914–924 (2002).

    Article  CAS  Google Scholar 

  61. Brewer, A.K. & Striegel, A.M. Characterizing the size, shape, and compactness of a polydisperse prolate ellipsoidal particle via quadruple-detector hydrodynamic chromatography. Analyst 136, 515–519 (2011).

    Article  CAS  PubMed  Google Scholar 

  62. Rubinson, K.A., Stanley, C. & Krueger, S. Small-angle neutron scattering and the errors in protein structures that arise from uncorrected background and intermolecular interactions. J. App. Crystallogr. 41, 456–465 (2008).

    Article  CAS  Google Scholar 

  63. Trewhella, J. et al. Report of the wwPDB Small-Angle Scattering Task Force: data requirements for biomolecular modeling and the PDB. Structure 21, 875–881 (2013).

    Article  CAS  PubMed  Google Scholar 

  64. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  PubMed  Google Scholar 

  65. Gasteiger, E. et al. Protein identification and analysis tools on the ExPASy server. in The Proteomics Protocols Handbook (ed. Walker, J.M.) 571–607 (Humana Press, 2005).

  66. Cleland,, W.W. Dithiothreitol, a new protective reagent for sh groups. Biochemistry 3, 480–482 (1964).

    Article  CAS  Google Scholar 

  67. Stevens, R., Stevens, L. & Price, N.C. The stabilities of various thiol compounds used in protein purifications. Biochem. Educ. 11, 70–70 (1983).

    Article  CAS  Google Scholar 

  68. Kreżel, A., Latajka, R., Bujacz, G.D. & Bal, W. Coordination properties of Tris(2-carboxyethyl)phosphine, a newly introduced thiol reductant, and its oxide. Inorg. Chem. 42, 1994–2003 (2003).

    Article  CAS  PubMed  Google Scholar 

  69. Krezel, A. et al. Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant. J. Inorg. Biochem. 84, 77–88 (2001).

    Article  CAS  Google Scholar 

  70. Han, J.C. & Han, G.Y. A procedure for quantitative determination of Tris(2-Carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal. Biochem. 220, 5–10 (1994).

    Article  CAS  PubMed  Google Scholar 

  71. Zhao, H., Brown, P.H. & Schuck, P. On the distribution of protein refractive index increments. Biophys. J. 100, 2309–2317 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Guinier, A. La diffraction des rayons X aux très pétits angles; application á l'etude de phénomènes ultramicroscopiques. Ann. Phys. 12, 161–237 (1939).

    Article  CAS  Google Scholar 

  73. 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 

  74. Glatter, O. A new method for the evaluation of small-angle scattering data. J. Appl. Crystallogr. 10, 415–421 (1977).

    Article  Google Scholar 

  75. 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 

  76. Krigbaum, W.R. & Kugler, F.R. Molecular conformation of egg-white lysozyme and bovine alpha-lactalbumin in solution. Biochemistry 9, 1216–1223 (1970).

    Article  CAS  PubMed  Google Scholar 

  77. Watson, M.C. & Curtis, J.E. Probing the average local structure of biomolecules using small-angle scattering and scaling laws. Biophys. J. 106, 2474–2482 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Fischer, H., de Oliveira Neto, M., 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 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Fritz, G. & Bergmann, A. SAXS instruments with slit collimation: investigation of resolution and flux. J. Appl. Crystallogr. 39, 64–71 (2006).

    Article  CAS  Google Scholar 

  81. 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 

  82. Voss, N.R. & Gerstein, M. Calculation of standard atomic volumes for RNA and comparison with proteins: RNA is packed more tightly. J. Mol. Biol. 346, 477–492 (2005).

    Article  CAS  PubMed  Google Scholar 

  83. 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 

  84. Wignall, G.D. & Bates, F.S. Absolute calibration of small-angle neutron scattering data. J. App. Crystallogr. 20, 28–40 (1987).

    Article  CAS  Google Scholar 

  85. Svergun, D.I. et al. Protein hydration in solution: experimental observation by x-ray and neutron scattering. Proc. Natl. Acad. Sci. USA 95, 2267–2272 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Leiting, B., Marsilio, F. & O'Connell, J.F. Predictable deuteration of recombinant proteins expressed in Escherichia coli. Anal. Biochem. 265, 351–355 (1998).

    Article  CAS  PubMed  Google Scholar 

  87. Goryunov, A.S. H/D isotope effects on protein hydration and interaction in solution. Gen. Physiol. Biophys. 25, 303–311 (2006).

    Article  CAS  PubMed  Google Scholar 

  88. Sheu, S.Y., Schlag, E.W., Selzle, H.L. & Yang, D.Y. Molecular dynamics of hydrogen bonds in protein-D2O: the solvent isotope effect. J. Phys. Chem. A 112, 797–802 (2008).

    Article  CAS  PubMed  Google Scholar 

  89. Baghurst, P.A., Nichol, L.W. & Sawyer, W.H. The effect of D 2 O on the association of -lactoglobulin A. J. Biol. Chem. 247, 3199–3204 (1972).

    CAS  PubMed  Google Scholar 

  90. Dougan, L., Koti, A.S., Genchev, G., Lu, H. & Fernandez, J.M. A single-molecule perspective on the role of solvent hydrogen bonds in protein folding and chemical reactions. Chemphyschem 9, 2836–2847 (2008).

    Article  CAS  PubMed  Google Scholar 

  91. Tehei, M., Madern, D., Pfister, C. & Zaccai, G. Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability. Proc. Natl. Acad. Sci. USA 98, 14356–14361 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Jasnin, M., Tehei, M., Moulin, M., Haertlein, M. & Zaccai, G. Solvent isotope effect on macromolecular dynamics in E. coli. Eur. Biophys. J. 37, 613–617 (2008).

    Article  CAS  PubMed  Google Scholar 

  93. Graewert, M.A. et al. Automated pipeline for purification, biophysical and x-ray analysis of biomacromolecular solutions. Sci. Rep. 5, 10734 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Glasoe, P.K. & Long, F.A. Use of glass electrodes to measure acidities in deuterium oxide. J. Phys. Chem. 64, 188–190 (1960).

    Article  CAS  Google Scholar 

  95. Svergun, D.I., Koch, M.H.J., Timmins, P.A. & May, R.P. Small Angle X-Ray and Neutron Scattering from Solutions of Biological Macromolecules. (Oxford University Press, 2013).

  96. Wood, K. et al. Exploring the structure of biological macromolecules in solution using Quokka, the small angle neutron scattering instrument, at ANSTO. Nucl. Instrum. Meth. A 798, 44–51 (2015).

    Article  CAS  Google Scholar 

  97. Mokbel, N. et al. K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity. Brain 136, 494–507 (2013).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  99. David, G. & Perez, J. Combined sampler robot and high-performance liquid chromatography: a fully automated system for biological small-angle X-ray scattering experiments at the Synchrotron SOLEIL SWING beamline. J. App. Crystallogr. 42, 892–900 (2009).

    Article  CAS  Google Scholar 

  100. Chen, X. et al. High yield expression and efficient purification of deuterated human protein galectin-2. Food Bioprod. Process. 90, 563–572 (2012).

    Article  CAS  Google Scholar 

  101. Sun, G. et al. Expression, purification and crystallization of human kynurenine aminotransferase 2 exploiting a highly optimized codon set. Protein Expr. Purif. 121, 41–45 (2016).

    Article  CAS  PubMed  Google Scholar 

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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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Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL) Hamburg Outstation, DESY, Hamburg, Germany

    Cy M Jeffries, Melissa A Graewert, Clément E Blanchet & Dmitri I Svergun

  2. Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia

    David B Langley

  3. Garvan Institute, Darlinghurst, New South Wales, Australia

    David B Langley

  4. Australian Centre for Neutron Science, ANSTO, Lucas Heights, New South Wales, Australia.,

    Andrew E Whitten

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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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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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