Cross-validation tests of time-averaged molecular dynamics refinements for determination of protein structures by X-ray crystallography.

Time-averaged structure-factor restraints have been used in two molecular dynamics refinement schemes to define ensembles of conformations for myoglobin that fit the experimentally measured Bragg scattering from P6 crystals. The geometries of the structures have been maintained to the same currently acceptable limits in all cases. Free R value analysis was used to assess the validity of the two approaches. In the first scheme, where atoms have no B values, the decrease in R value was found to be spurious as judged by a concomitant increase in the free R value. The other scheme, however, which retains individual B values, was found to yield both low R values and low free R values; thus, here the additional variables introduced by modeling the protein in terms of an evolving ensemble of states do not overfit the data. For comparison, refinements were also carried out on the system using several other techniques for isotropic and anisotropic crystallographic refinement. The time-averaged refinements with B values compare quite favorably with the standard methods, but yield additional information about substates of the system. Hence, correctly applied time-averaged refinements can yield accurate models for protein molecules; moreover, by essentially relaxing the harmonic approximation from the refinement process, these refinements allow a more detailed description of the motions of complex molecules, such as proteins, to be determined from X-ray crystallographic data.