Toward ab initio refinement of protein X-ray crystal structures: interpreting and correlating structural fluctuations |
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Authors: | Olle?Falkl?f Charles?A?Collyer Email author" target="_blank">Jeffrey?R?ReimersEmail author |
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Institution: | 1.School of Chemistry,The University of Sydney,Sydney,Australia;2.Department of Chemistry,The University of Gothenburg,Gothenburg,Sweden;3.School of Molecular Bioscience,The University of Sydney,Sydney,Australia;4.Department of Physics, Chemistry and Biology,Link?ping University,Link?ping,Sweden |
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Abstract: | The refinement of protein crystal structures currently involves the use of empirical restraints and force fields that are
known to work well in many situations but nevertheless yield structural models with some features that are inconsistent with
detailed chemical analysis and therefore warrant further improvement. Ab initio electronic structure computational methods
have now advanced to the point at which they can deliver reliable results for macromolecules in realistic times using linear-scaling
algorithms. The replacement of empirical force fields with ab initio methods in a final refinement stage could allow new structural
features to be identified in complex structures, reduce errors and remove computational bias from structural models. In contrast
to empirical approaches, ab initio refinements can only be performed on models that obey basic qualitative chemical rules,
imposing constraints on the parameter space of existing refinements, and this in turn inhibits the inclusion of unlikely structural
features. Here, we focus on methods for determining an appropriate ensemble of initial structural models for an ab initio
X-ray refinement, modeling as an example the high-resolution single-crystal X-ray diffraction data reported for the structure
of lysozyme (PDB entry “2VB1”). The AMBER force field is used in a Monte Carlo calculation to determine an ensemble of 8 structures
that together embody all of the partial atomic occupancies noted in the original refinement, correlating these variations
into a set of feasible chemical structures while simultaneously retaining consistency with the X-ray diffraction data. Subsequent
analysis of these results strongly suggests that the occupancies in the empirically refined model are inconsistent with protein
energetic considerations, thus depicting the 2VB1 structure as a deep-lying minimum in its optimized parameter space that
actually embodies chemically unreasonable features. Indeed, density-functional theory calculations for one specific nitrate
ion with an occupancy of 62% indicate that water replaces this ion 38% of the time, a result confirmed by subsequent crystallographic
analysis. It is foreseeable that any subsequent ab initio refinement of the whole structure would need to locate a globally improved structure involving significant changes to 2VB1 which correct these identified local structural inconsistencies. |
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