The prevalence of problematic geometries observed in deposited PDB structures suggests that conventional refinement methods are not sufficiently rigorous to represent the chemistry within the protein–ligand complex (Kleywegt, 2007 Pozharski et al., 2013 Smart et al., 2018 ). Furthermore, this median score deteriorates as the resolution decreases (Read et al., 2011 ). In particular, the median clashscore, which is the number of clashes per 1000 atoms, for all X-ray structures deposited in the Protein Data Bank (PDB) and the worldwide PDB (wwPDB) (Berman et al., 2003, 2007 ) since 1990, and determined at a resolution of 1.5 Å or better, is 8.8 units. This has led to the development of structure-validation metrics, including Ramachandran, clashscore and MolProbity score, the latter of which is a composite of the clashscore and Ramachandran plot and rotamer outliers (Ramachandran et al., 2011 Read et al., 2011 Chen et al., 2010 MacCallum et al., 2009 ). However, protein crystal models are still subject to significant uncertainties in atomic coordinates and other structural errors (Davis et al., 2003, 2007 ), and these errors negatively impact the very ligand-binding affinity estimations (Davis et al., 2003 ) that are critical to SBDD/FBDD applications. In recent years, X-ray crystallography has become routine thanks to advances in data collection and processing, structure solution and refinement automation. The quality of the model is crucial for the overall success of high-throughput screening, docking and scoring (for example rank ordering) of potential drug candidates. X-ray crystallography is a popular technique that is used to determine the three-dimensional atomic structures of biomolecular systems, which serve as three-dimensional templates for structure-based drug discovery (SBDD) and fragment-based drug discovery (FBDD). Overall, these results demonstrate that the use of a structure-wide QM/MM Hamiltonian exhibits improvements in the local structural chemistry of the ligand similar to Region-QM refinement but with significant improvements in the overall structure beyond the active site. Across the entire population, this method results in an average 3.5-fold reduction in ligand strain and a 4.5-fold improvement in MolProbity clashscore, as well as improvements in Ramachandran and rotamer outlier analyses. This approach was validated through the automatic treatment of a population of 80 protein–ligand structures chosen from the Astex Diverse Set. In the present work, this approach is expanded to include a more rigorous treatment of the entire structure, including the ligand(s), the associated active site(s) and the entire protein, using a fully automated, mixed quantum-mechanics/molecular-mechanics (QM/MM) Hamiltonian recently implemented in the DivCon package. This method (Region-QM) significantly increases the accuracy of the X-ray refinement process, and this approach is now used, coupled with experimental density, to accurately determine protonation states, binding modes, ring-flip states, water positions and so on. Recently, plugins to the Python-based Hierarchical ENvironment for Integrated Xtallography ( PHENIX) crystallographic platform have been developed to augment conventional methods with the in situ use of quantum mechanics (QM) applied to ligand(s) along with the surrounding active site(s) at each step of refinement. While this approach has served the crystallographic community for decades, as structure-based drug design/discovery (SBDD) has grown in prominence it has become clear that these conventional methods are less rigorous than they need to be in order to produce properly predictive protein–ligand models, and that the human intervention that is required to successfully treat ligands and other unusual chemistries found in SBDD often precludes high-throughput, automated refinement. Conventional macromolecular crystallographic refinement relies on often dubious stereochemical restraints, the preparation of which often requires human validation for unusual species, and on rudimentary energy functionals that are devoid of nonbonding effects owing to electrostatics, polarization, charge transfer or even hydrogen bonding.
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