On the application of molecular-dynamics simulations to validate thermal parameters and to optimize TLS-group selection for macromolecular refinement

Comparison of crystallographically determined and molecular dynamics simulation-derived parameters for a small (26 kDa) homotetrameric four-α-helical bundle protein revealed an unexpected pattern of similarities and differences between experiment and simulation. On one hand, the protein structure per se is exceptionally well preserved during the simulations, with a root-mean-square deviation between the C^α atoms of the crystal structure and the simulation-derived average structures of only 0.58 Å, which is not very different from the expected coordinate error of the experimentally determined structure. On the other hand, comparison of the temperature factors showed a large discrepancy, with the experimental B factors being approximately three times higher than the simulation-derived B factors. Closer examination of this discrepancy appears to validate the molecular-dynamics prediction and to implicate as its source static disorder at the crystalline state, as indicated by the strong diffuse scattering and pronounced anisotropy of the diffraction pattern of the protein crystals. A posteriori re-refinement of the structure using a new TLS parameterization scheme based on the results obtained from the simulations led to a further reduction of the R factor and the free R value by 0.4% and 0.8%, respectively, indicating that molecular-dynamics simulations have matured to the point that they can be used to aid the selection of TLS groups for macromolecular refinement.