Small-angle scattering for structural biology--expanding the frontier while avoiding the pitfalls

Abstract

The last decade has seen a dramatic increase in the use of small-angle scattering for the study of biological macromolecules in solution. The drive for more complete structural characterization of proteins and their interactions, coupled with the increasing availability of instrumentation and easy-to-use software for data analysis and interpretation, is expanding the utility of the technique beyond the domain of the biophysicist and into the realm of the protein scientist. However, the absence of publication standards and the ease with which 3D models can be calculated against the inherently 1D scattering data means that an understanding of sample quality, data quality, and modeling assumptions is essential to have confidence in the results. This review is intended to provide a road map through the small-angle scattering experiment, while also providing a set of guidelines for the critical evaluation of scattering data. Examples of current best practice are given that also demonstrate the power of the technique to advance our understanding of protein structure and function.

Figure 1

Roadmap through the small-angle scattering experiment.

Figure 2

Small-angle scattering from a complex between a deuterated and nondeuterated protein: the contrast variation experiment. The figure illustrates contrast variation using a structure (PDB: 3GMR) of T-cell surface glycoprotein CD1d1 in complex with beta-2 microglobulin. Theoretical scattering profiles were generated for the protein complex in which the beta-2 microglobulin component had its nonexchangeable hydrogens deuterated to 60% in silico. (A) The proteins each have the same electron density and hence X-ray scattering density and the small-angle X-ray scattering profile therefore yield information on the shape of the entire complex as a uniform contrast (depicted as all black) particle. (B) The neutron scattering contrast for the deuterated (90% gray) and nondeuterated (40% gray) components is distinct, and it is therefore possible to measure scattering from each individual component by solvent matching the scattering density of the other by H/D substitution in the solvent. The nondeuterated protein will be solvent matched around 40% D₂O, whereas the deuterated protein's solvent match point will be 90% D₂O, this value depending upon the deuteration level of the protein (60% in this example). (C) A theoretical set of data acquired for different %D₂O in the solvent (a contrast series) yields information on the shapes and dispositions of the deuterated and nondeuterated proteins. From the contrast series, it is possible to extract scattering profiles corresponding to the deuterated and nondeuterated components (red and blue, respectively) and a cross-term (green) that is related to their relative dispositions. Note that because of the smaller size of the deuterated component, the corresponding P(r) needs to be multiplied by a factor of five to observe the curve clearly. Also, the cross-term contains negative values for I(q) and therefore must be plotted on a linear scale.

Figure 3

Effects of interparticle interference and aggregation on small-angle scattering data. It is often difficult to assess sample quality from I(q) plots alone (A–C). Guinier plots (D–F) can be instructive, as deviations from linearity are clear indicators of interparticle interference (E, orange) or aggregation (F, red). The P(r) presented with a single chosen D_max (G–I) can be deceptive, and it is possible that only severe aggregation is detected (I, red). Behavior of the P(r) at extended D_max values (J–L) gives the clearest indication of sample quality. It is important to note that the subtle cases (K, dark yellow and L, magenta) may escape detection by these initial data inspection methods, and as such comparison of the data to secondary standards is essential. In evaluating these differences of around 1 Å in R_g, it is important to note that such a difference is well outside the precision with which R_g can be determined (generally to within a few tenths of Å for a small- to medium-sized protein), and that this degree of inaccuracy will significantly bias any 3D modeling performed against the scattering data.