Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

Abstract

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein-solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.diffuse scattering | protein crystallography | molecular-dynamics simulation | protein dynamics | staphylococcal nuclease P roteins explore many conformations while carrying out their functions in biological systems (1-3). X-ray crystallography is the dominant source of information about protein structure; however, crystal structure models usually consist of just a single major conformation and at most a small portion of the model as alternate conformations. Crystal structures therefore are missing many details about the underlying conformational ensemble (4).Proteins assembled in crystalline arrays, like proteins in solution, exhibit rich conformational diversity (4) and often can perform their native functions (5). Many methods have emerged for using Bragg data to model conformational diversity in protein crystals (6-17). The development of these methods has been important as conformational diversity can lead to inaccuracies in protein structure models (9,(18)(19)(20). A key limitation of using the Bragg data, however, is that different models of conformational diversity can yield the same mean electron density.Whereas the Bragg scattering only contains information about the mean electron density, diffuse scattering (diffraction resulting in intensity between the Bragg peaks) is sensitive to spatial correlations in electron density variations (21-28) and therefore contains information about the way that atomic positions vary together in protein crystals. Because models that yield the same mean electron density c...

Measuring and modeling diffuse scattering in protein X-ray crystallography

“…In particular, MD simulations now provide sufficient sampling to yield robust calculations of diffuse intensity (13), and these can be used to consider a myriad of intramolecular motions (e.g., loop openings and side-chain flips) (58) and lattice dynamics. Polikanov and Moore (46) recently have demonstrated the importance of lattice vibrations in explaining experimental diffuse scattering measurements of ribosome crystals, which indicates that models should simultaneously account for correlations that are coupled both within and across unit cell boundaries (18,20); accounting for lattice vibrations more accurately also might yield improved Bragg integration (48).…”

“…Experimental and model diffuse intensities were compared using just the anisotropic component of the signal, which is primarily due to the protein (13). Lunus methods were used to subtract the radial average and obtain the anisotropic signal.…”

“…There is also a long-standing interest both in using diffuse scattering to validate improvements in MD simulations and in using MD to derive a structural basis for the protein motions that give rise to diffuse scattering (13,31,(34)(35)(36)(37)(38)(39). Recent advances in computing now enable microsecond duration simulations (13) that can overcome past barriers to accurate calculations seen using 10-ns or shorter MD trajectories (35,38).…”

“…Time-averaged ensemble refinement (11) is another possibility, but it is complicated by the use of a TLS model to account for crystal packing variations (11). Solid-state NMR experiments (12) and long-timescale molecular dynamics (MD) simulations (13)(14)(15) can be used to probe the structural basis of crystal packing variations and internal protein motions.…”

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

“…Recent publications have provided direct evidence that supports the notion of the validity of the universal model for the crystalline proteins. Wall et al (2014) investigated the conformational dynamics of a crystalline protein with the use of accurately measured scattered X-ray diffraction obtained from protein crystals and used MD simulations to analyze the dynamics of the molecules in the crystal phase. They found eight metastable states of the structure of staphylococcal nuclease extending from 4 ns up to 1100 ns.…”