Abstract
Biomolecular X-ray structures typically provide a static, time- and ensemble-averaged view of molecular ensembles in crystals. In the absence of rigid-body motions and lattice defects, B-factors are thought to accurately reflect the structural heterogeneity of such ensembles. In order to study the effects of averaging on B-factors, we employ molecular dynamics simulations to controllably manipulate microscopic heterogeneity of a crystal containing 216 copies of villin headpiece. Using average structure factors derived from simulation, we analyse how well this heterogeneity is captured by high-resolution molecular-replacement-based model refinement. We find that both isotropic and anisotropic refined B-factors often significantly deviate from their actual values known from simulation: even at high 1.0 Å resolution and Rfree of 5.9%, B-factors of some well-resolved atoms underestimate their actual values even sixfold. Our results suggest that conformational averaging and inadequate treatment of correlated motion considerably influence estimation of microscopic heterogeneity via B-factors, and invite caution in their interpretation.
Highlights
Biomolecular X-ray structures typically provide a static, time- and ensemble-averaged view of molecular ensembles in crystals
While these effects have been well studied in the area of biomolecular nuclear magnetic resonance (NMR) and other spectroscopic methods, they are still largely underexplored when it comes to scattering experiments with some exceptions[4,9,10,11,12,13]
With a total of 27 unit cells, 216 protein copies and a total of 118,752 atoms including solvent, the villin headpiece crystal studied here is to the best of our knowledge one of the largest biomolecular crystals simulated to date using atomistic MD (Fig. 1a)
Summary
Biomolecular X-ray structures typically provide a static, time- and ensemble-averaged view of molecular ensembles in crystals. Dynamics directly influences the very process of structure determination as structural averages derived from diverse ensembles may exhibit certain features, which are not necessarily representative of the true microscopic reality and are a direct artifact of averaging[4,8,9,10] While these effects have been well studied in the area of biomolecular nuclear magnetic resonance (NMR) and other spectroscopic methods, they are still largely underexplored when it comes to scattering experiments with some exceptions[4,9,10,11,12,13]. We analyse the resultant model and compare the refined harmonic isotropic or anisotropic B-factors against their MD-based counterparts, which in turn represent atomic fluctuations that are present in the system used for the generation of structure factors Such internally self-consistent comparison between the refined model and the actual microscopic ensemble known from the simulation allows us to study how accurately X-ray refinement captures the true structural diversity of a biomolecule present at the microscopic level
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