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An optimized SEC‐SAXS system enabling high X‐ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis
Author(s) -
Ryan Timothy M.,
Trewhella Jill,
Murphy James M.,
Keown Jeremy R.,
Casey Lachlan,
Pearce F. Grant,
Goldstone David C.,
Chen Kelan,
Luo Zhenyao,
Kobe Bostjan,
McDevitt Christopher A.,
Watkin Serena A.,
Hawley Adrian M.,
Mudie Stephen T.,
Samardzic Boban Vesna,
Kirby Nigel
Publication year - 2018
Publication title -
journal of applied crystallography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.429
H-Index - 162
ISSN - 1600-5767
DOI - 10.1107/s1600576717017101
Subject(s) - small angle x ray scattering , beamline , scattering , synchrotron , materials science , synchrotron radiation , analytical chemistry (journal) , chemistry , optics , chromatography , beam (structure) , physics
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.