Molecular dynamics simulations of crystals can enlighten interpretation of experimental X-ray crystallography data and elucidate structural dynamics and heterogeneity in biomolecular crystals. Furthermore, because of the direct comparison against experimental data, they can inform assessment of molecular dynamics methods and force fields. We present microsecond scale results for triclinic hen egg-white lysozyme in a supercell consisting of 12 independent unit cells using four contemporary force fields (Amber ff99SB, ff14ipq, ff14SB, and CHARMM 36) in crystalline and solvated states (for ff14SB only). We find the crystal simulations consistent across multiple runs of the same force field and robust to various solvent equilibration schemes. However, convergence is slow compared with solvent simulations. All the tested force fields reproduce experimental structural and dynamic properties well, but Amber ff14SB maintains structure and reproduces fluctuations closest to the experimental model: its average backbone structure differs from the deposited structure by 0.37Å ; by contrast, the average backbone structure in solution differs from the deposited by 0.65Å . All the simulations are affected by a small progressive deterioration of the crystal lattice, presumably due to imperfect modeling of hydrogen bonding and other crystal contact interactions; this artifact is smallest in ff14SB, with average lattice positions deviating by 0.20Å from ideal. Side-chain disorder is surprisingly low with fewer than 30% of the nonglycine or alanine residues exhibiting significantly populated alternate rotamers. Our results provide helpful insight into the methodology of biomolecular crystal simulations and indicate directions for future work to obtain more accurate energy models for molecular dynamics.

The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. Residues A38 and G8 have been implicated as playing roles in general acid and base catalysis, respectively. Here we explore the structure and dynamics of key active site residues using more than 1 μs of molecular dynamics simulations of the hairpin ribozyme at different stages along the catalytic pathway. Analysis of results indicates hydrogen bond interactions between the nucleophile and proR nonbridging oxygen are correlated with active inline attack conformations. Further, the simulation results suggest a possible alternative role for G8 to promote inline fitness and facilitate activation of the nucleophile by hydrogen bonding, although this does not necessarily exclude an additional role as a general base. Finally, we suggest that substitution of G8 with N7- or N3-deazaguanosine which have elevated pKa values, both with and without thio modifications at the 5′ leaving group position, would provide valuable insight into the specific role of G8 in catalysis.

Molecular dynamics simulations of biomolecular crystals at atomic resolution have the potential to recover information on dynamics and heterogeneity hidden in the X-ray diffraction data. We present here 9.6 microseconds of dynamics in a small helical peptide crystal with 36 independent copies of the unit cell. The average simulation structure agrees with experiment to within 0.28 Å backbone and 0.42 Å all-atom rmsd; a model refined against the average simulation density agrees with the experimental structure to within 0.20 Å backbone and 0.33 Å all-atom rmsd. The R-factor between the experimental structure factors and those derived from this unrestrained simulation is 23% to 1.0 Å resolution. The B-factors for most heavy atoms agree well with experiment (Pearson correlation of 0.90), but B-factors obtained by refinement against the average simulation density underestimate the coordinate fluctuations in the underlying simulation where the simulation samples alternate conformations. A dynamic flow of water molecules through channels within the crystal lattice is observed, yet the average water density is in remarkable agreement with experiment. A minor population of unit cells is characterized by reduced water content, 310 helical propensity and a gauche(−) side-chain rotamer for one of the valine residues. Careful examination of the experimental data suggests that transitions of the helices are a simulation artifact, although there is indeed evidence for alternate valine conformers and variable water content. This study highlights the potential for crystal simulations to detect dynamics and heterogeneity in experimental diffraction data, as well as to validate computational chemistry methods.

Modern crystal structure refinement programs rely on geometry restraints to overcome the challenge of a low data-to-parameter ratio. While the classical Engh and Huber restraints work well for standard amino-acid residues, the chemical complexity of small-molecule ligands presents a particular challenge. Most current approaches either limit ligand restraints to those that can be readily described in the Crystallographic Information File (CIF) format, thus sacrificing chemical flexibility and energetic accuracy, or they employ protocols that substantially lengthen the refinement time, potentially hindering rapid automated refinement workflows. PHENIX-AFITT refinement uses a full molecular-mechanics force field for user-selected small-molecule ligands during refinement, eliminating the potentially difficult problem of finding or generating high-quality geometry restraints. It is fully integrated with a standard refinement protocol and requires practically no additional steps from the user, making it ideal for high-throughput workflows. PHENIX-AFITT refinements also handle multiple ligands in a single model, alternate conformations and covalently bound ligands. Here, the results of combining AFITT and the PHENIX software suite on a data set of 189 protein-ligand PDB structures are presented. Refinements using PHENIX-AFITT significantly reduce ligand conformational energy and lead to improved geometries without detriment to the fit to the experimental data. For the data presented, PHENIX-AFITT refinements result in more chemically accurate models for small-molecule ligands.