Predicting data quality in biological X-ray solution scattering

Abstract

Biological small-angle X-ray solution scattering (BioSAXS) is now widely used to gain information on biomolecules in the solution state. Often, however, it is not obvious in advance whether a particular sample will scatter strongly enough to give useful data to draw conclusions under practically achievable solution conditions. Conformational changes that appear to be large may not always produce scattering curves that are distinguishable from each other at realistic concentrations and exposure times. Emerging technologies such as time-resolved SAXS (TR-SAXS) pose additional challenges owing to small beams and short sample path lengths. Beamline optics vary in brilliance and degree of background scatter, and major upgrades and improvements to sources promise to expand the reach of these methods. Computations are developed to estimate BioSAXS sample intensity at a more detailed level than previous approaches, taking into account flux, energy, sample thickness, window material, instrumental background, detector efficiency, solution conditions and other parameters. The results are validated with calibrated experiments using standard proteins on four different beamlines with various fluxes, energies and configurations. The ability of BioSAXS to statistically distinguish a variety of conformational movements under continuous-flow time-resolved conditions is then computed on a set of matched structure pairs drawn from the Database of Macromolecular Motions (http://molmovdb.org). The feasibility of experiments is ranked according to sample consumption, a quantity that varies by over two orders of magnitude for the set of structures. In addition to photon flux, the calculations suggest that window scattering and choice of wavelength are also important factors given the short sample path lengths common in such setups.

High-pressure small-angle X-ray scattering cell for biological solutions and soft materials

“…It is most likely that this transient scale shift observed in the initial 10 MPa data comes from the plastic sample-cell windows. The scale differences between ambient and high pressure can be attributed to changes in contrast between the protein and buffer: I(0) / c 2 ( protein À buffer ) 2 , where c = sample concentration, = protein specific volume and = bulk electron density (3.3 Â 10 21 cm À3 for water and 4.4 Â 10 21 cm À3 for protein) (Ando et al, 2008;Wang et al, 2018). Since the observed protein compressibilities are often small in comparison with water, we will assume that only the water volume changes (Grindley & Lind, 1971;Kharakoz, 2000).…”

“…SAXS from biological solutions (BioSAXS) is dependent on measuring very small changes in the SAXS signals between a reference solution and one containing the macromolecules of interest, both of which scatter weakly (Skou et al, 2014). In consequence, it is of the utmost importance that all sources of background scatter be reduced as much as possible and remain reproducible when the reference solution is substituted for the sample solution (Wang et al, 2018). The need to minimize background scatter dictated a vacuum enclosure that included the downstream HP-SAXS cell window and the detector.…”

Use of continuous sample translation to reduce radiation damage for XPCS studies of protein diffusion

“…Radiation damage to samples is of critical importance in the X-ray and electron microscopy communities and a number of works have reviewed both the mechanisms of radiation damage and experimental strategies to reduce such damage (Wang et al, 2018;Garman & Weik, 2017;Costa et al, 2016;Ueno et al, 2019;Garrison, 1987;Meisburger et al, 2013;Egerton, 2019;Polsinelli et al, 2017;Hopkins & Thorne, 2016 Massover, 2007;Kirby et al, 2016). Radiation damage in protein samples has been studied by the macromolecular crystallography (MX) and small-angle X-ray scattering (SAXS) communities.…”