Observing the overall rocking motion of a protein in a crystal

Abstract

The large majority of three-dimensional structures of biological macromolecules have been determined by X-ray diffraction of crystalline samples. High-resolution structure determination crucially depends on the homogeneity of the protein crystal. Overall ‘rocking' motion of molecules in the crystal is expected to influence diffraction quality, and such motion may therefore affect the process of solving crystal structures. Yet, so far overall molecular motion has not directly been observed in protein crystals, and the timescale of such dynamics remains unclear. Here we use solid-state NMR, X-ray diffraction methods and μs-long molecular dynamics simulations to directly characterize the rigid-body motion of a protein in different crystal forms. For ubiquitin crystals investigated in this study we determine the range of possible correlation times of rocking motion, 0.1–100 μs. The amplitude of rocking varies from one crystal form to another and is correlated with the resolution obtainable in X-ray diffraction experiments.

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

“…Interestingly, this finding mirrors the picture provided by the comparison between solution and crystal data ( Figure 34): also there, differences in motions were found for exactly the same regions, while the majority of the protein had very similar motional parameters. MD simulations on different crystal forms of ubiquitin support this view [183,189]. The picture that emerges from the various comparisons above is, therefore, that the crystalline environment does not detectably alter the dynamics on sub-microsecond time scales, except for a few residues located in loop regions.…”

“…The rocking motion should also lead to slightly lower order parameters, and also this observation is experimentally found (see panel b). Additional support for such overall "rocking" motion in the crystal comes from MD simulations of the different crystals, which detects significant overall residual motion for cubic-PEG-ub [189]. It may be that such overall motion, found here in one particular crystal form, is a general contribution to resolution of solid-state NMR spectra.…”

“…Panel (e) provides evidence that such a restricted overall motion also has consequences for X-ray observables: cubic-PEG-ub crystals diffract to lower resolution, and the Wilson-B factor is higher than in the other two crystal forms (two independent data sets are reported for the PEG-derived crystals). Reproduced with permission from reference [189]. Conformational exchange processes on μs time scales in ubiquitin in solution and crystals.…”

“…have studied dynamics of ubiquitin in three different crystal forms (crystallizing in different space groups) [189]. Figure 36 compares (panels a and b) observables that are primarily sensitive to sub-microsecond motions.…”

Protein crystal lattices are dynamic assemblies: the role of conformational entropy in the protein condensed phase

“…They found eight metastable states of the structure of staphylococcal nuclease extending from 4 ns up to 1100 ns. Ma et al (2015) observed rocking motions in the structure of ubiquitin crystallized in different morphologies. Using three techniques, X-ray diffraction, solid-state NMR and MD simulations, they found that the three-dimensional structure of ubiquitin oscillated on a time scale of 0.1 ms to 100 ms.…”