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Type: Journal article
Title: An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis
Author: Ryan, T.
Trewhella, J.
Murphy, J.
Keown, J.
Casey, L.
Pearce, F.
Goldstone, D.
Chen, K.
Luo, Z.
Kobe, B.
McDevitt, C.
Watkin, S.
Hawley, A.
Mudie, S.
Samardzic Boban, V.
Kirby, N.
Citation: Journal of Applied Crystallography, 2018; 51(1):97-111
Publisher: International Union of Crystallography
Issue Date: 2018
ISSN: 0021-8898
Statement of
Timothy M. Ryan, Jill Trewhella, James M. Murphy, Jeremy R. Keown, Lachlan Casey, F. Grant Pearce, David C. Goldstone, Kelan Chen, Zhenyao Luo, Bostjan Kobe, Christopher A. McDevitt, Serena A. Watkin, Adrian M. Hawley, Stephen T. Mudie, Vesna Samardzic Boban and Nigel Kirby
Abstract: A new optimized size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) system for biomolecular SAXS at the Australian Synchrotron SAXS/WAXS beamline has been developed. The compact configuration reduces sample dilution to maximize sensitivity. Coflow sample presentation allows an 11-fold increase in flux on sample without capillary fouling, improving throughput and data quality, which are now primarily limited by the full flux available on the beamline. Multi-wavelength fibre optic UV analysis in close proximity to the X-ray beam allows for accurate concentration determination for samples with known UV extinction coefficients and thus estimation of the molecular weight of the scattering particle from the forward X-ray scattering intensity. Fast-flow low-volume SEC columns provide sample throughput competitive with batch concentration series measurements, albeit with a concomitant reduction of potential resolution relative to lower flow rates and larger SEC columns. The performance of the system is demonstrated using a set of model proteins, and its utility to solve various challenges is illustrated with a diverse suite of protein samples. These developments increase the quality and rigor of SEC-SAXS analysis and open new avenues for biomolecular solution SEC-SAXS studies that have been challenged by low sample yields, temporal instability, radiation sensitivity and complex mixtures.
Keywords: Size exclusion chromatography; small-angle X-ray scattering; SEC-SAXS; coflow; SAXS; polydisperse proteins; molecular weight
Rights: © 2018 International Union of Crystallography
RMID: 0030081792
DOI: 10.1107/S1600576717017101
Grant ID:
Appears in Collections:Molecular and Biomedical Science publications

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