Synchrotron-based small-angle X-ray scattering of proteins in solution

Abstract

Summary With recent advances in data analysis algorithms, X-ray detectors, and synchrotron sources, small-angle X-ray scattering (SAXS) has become much more accessible to the structural biology community than ever before. Although limited to ~10 Å resolution, SAXS can provide a wealth of structural information on biomolecules in solution and is compatible with a wide range of experimental conditions. SAXS is thus an attractive alternative when crystallography is not possible. Moreover, advanced usage of SAXS can provide unique insight into biomolecular behavior that can only be observed in solution, such as large conformational changes and transient protein-protein interactions. Unlike crystal diffraction data, however, solution scattering data are subtle in appearance, highly sensitive to sample quality and experimental errors, and easily misinterpreted. In addition, synchrotron beamlines that are dedicated to SAXS are often unfamiliar to the non-specialist. Here, we present a series of procedures that can be used for SAXS data collection and basic cross-checks designed to detect and avoid aggregation, concentration effects, radiation damage, buffer mismatch, and other common problems. The protein, human serum albumin (HSA), serves as a convenient and easily replicated example of just how subtle these problems can sometimes be, but also of how proper technique can yield pristine data even in problematic cases. Because typical data collection times at a synchrotron are only one to several days, we recommend that the sample purity, homogeneity, and solubility be extensively optimized prior to the experiment.

Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions

“…Scattering patterns I(q) are also shown as Kratky plots (q 2 I(q) vs q, Figure S2A) indicating that all proteins are properly folded in solution. Typically, a globular, structured protein exhibits a pronounced maximum (bell-shaped curve), whereas a random chain (for example, an unfolded protein) will plateau (47,48). Then, Porod-Debye plots (q 4 I(q) vs. q 4 ) were generated and confirmed the compactness of all proteins ( Figure S2B and Table S2).…”

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules

“…It was found that even at low dose (< 0.5 Gy) chromatin shows radiation damage as evidence by a change in hydrodynamic size that was likely due to single-strand breaks in DNA [58]. In SAXS, radiation damage most often present itself as aggregation [59]. Even with sample flow enable, radiation damage in lysozyme, evidenced by an increase in radius of gyration ( R g ), still occurs as early as 250 ms exposure time [11].…”

Monomeric green fluorescent protein as a protein standard for small angle scattering

“…A number of proteins have been used as calibration standards, the four main ones being: i) lysozyme [6], ii) bovine (BSA) [6,8] or human serum albumin (HSA) [9] and iii) glucose isomerase [5]. For proteins to be considered as a calibration standard they need a number of attributes, specifically being; i) cheap and abundantly available, ii) monodisperse and iii) thermodynamically stable over time, so giving reproducible and expected results, most commonly using Guinier analysis to determine results such as radius of gyration (Rg) and intensity at zero angle I(0) [4,10].…”