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, TM; Trewhella, J; Murphy, JM; Keown, JR; Casey, L; Pearce, FG; Goldstone, DC; Chen, K; Luo, Z; Kobe, B; McDevitt, CA; Watkin, SA; Hawley, AM; Mudie, ST; Samardzic Boban, V; Kirby, N;
- Details
- Publication Year 2018,Volume 51,Issue #1,Page 97-111
- Journal Title
- Journal of Applied Crystallography
- Publication Type
- Journal Article
- 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.Size exclusion chromatography (SEC) small-angle X-ray scattering (SAXS) is a powerful structural biology tool where the best outcomes are obtained through an optimized experimental approach. Optimization of SEC and integration of a sheath flow sample environment into the Australian Synchrotron's SAXS/WAXS beamline has greatly improved data quality and the ability to deal with difficult protein samples. © International Union of Crystallography, 2018.
- Publisher
- Wiley-Blackwell
- Research Division(s)
- Cell Signalling And Cell Death
- Publisher's Version
- https://doi.org/10.1107/S1600576717017101
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2018-03-19 10:02:59
Last Modified: 2018-03-19 12:28:37