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Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

Abstract

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

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Figure 1: High-throughput SAXS pipeline.
Figure 2: SAXS analysis provides feedback on 'challenging' samples that are polydisperse or inhomogeneous.
Figure 3: SAXS provides accurate shape and assembly in solution for most samples.
Figure 4: SAXS determines accurate assembly state in solution, as shown for acetyl-CoA synthetase subunit (PF1787).
Figure 5: SAXS defines accurate shape and assembly in solution for unknown structures and can uncover unsuspected structural similarity.

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Acknowledgements

This research is part of the Molecular Assemblies: Genes and Genomes Integrated Efficiently (MAGGIE) project supported by the US Department of Energy (DOE; DE-FG0207ER64326) and benefited from allocation of supercomputer time at the National Energy Research Scientific Computing Center (NERSC). Support for advancement of SAXS technologies at the Lawrence Berkeley National Laboratory SIBYLS beamline of the Advanced Light Source came from the DOE program Integrated Diffraction Analysis Technologies (IDAT) under contract DE-AC02-05CH11231 with the DOE. We thank I. Wilson and M. Knuth (the Scripps Research Institute) for providing protein samples from their Joint Center for Structural Genomics (JCSG).

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Contributions

G.L.H., J.A.T., M.H., S.C. and K.A.F. designed the SIBYLS beamline for high throughput. G.L.H., J.A.T. and M.W.W.A. wrote the manuscript. A.L.M., F.L.P., F.E.J., S.E.T., R.P.R., R.C.H. and G.L.H. prepared samples for data collection. G.L.H. and S.E.T. collected SAXS data. M.H. and G.L.H. wrote code for analysis. R.P.R. designed http://www.bioisis.net/. S.-j.Y. prepared PF0380 and PF2047.1. B.D.D. prepared PF0014/0015. J.W.S. prepared PF1787.

Corresponding authors

Correspondence to Michael W W Adams or John A Tainer.

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Hura, G., Menon, A., Hammel, M. et al. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS). Nat Methods 6, 606–612 (2009). https://doi.org/10.1038/nmeth.1353

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