Bringing diffuse X-ray scattering into focus

Abstract

X-ray crystallography is experiencing a renaissance as a method for probing the protein conformational ensemble. The inherent limitations of Bragg analysis, however, which only reveals the mean structure, have given way to a surge in interest in diffuse scattering, which is caused by structure variations. Diffuse scattering is present in all macromolecular crystallography experiments. Recent studies are shedding light on the origins of diffuse scattering in protein crystallography, and provide clues for leveraging diffuse scattering to model protein motions with atomic detail.

Figure 1

A typical detector image in X-ray crystallography (from [19^••]) (upper, left) records all of the X-rays scattered by a protein crystal during a single exposure. Dark pixels correspond to high X-ray intensities. A cartoon crystal is depicted (lower, left) that contains a series of unit cells, with the contents of any given unit cell adopting one of two conformations (the conformations are expected to be more varied in a real protein crystal). Conformation A is shown in orange, while conformation B is shown in black (lower panel). During analysis, data are reduced by examining only the Bragg peaks (upper, middle), which report on the average charge density within a unit cell (lower, middle). The electron density is shown in blue, with areas of especially strong charge highlighted in purple. While multiple conformations may be modeled into the average density, assigning which conformations occur together across residues requires additional information. Current modeling practices use geometric constraints to help classify different alternative conformation groups. The diffuse scattering left behind during data reduction (upper, right) is an additional potential source of such information. Diffuse scattering includes an isotropic component that is determined both by protein and solvent scattering [21,22^••], and an anisotropic component that is dominated by correlated protein motions within the crystal [22^••]. Analyzing this anisotropic signal might help to distinguish networks of residues that move together (lower, right).

Figure 2

Comparison of simulated diffuse intensity in diffraction images computed from (a) a refined ENM of staphylococcal nuclease and (b) experimental data from Ref. [18].