Accurate macromolecular crystallographic refinement: incorporation of the linear scaling, semiempirical quantum-mechanics program DivCon into the PHENIX refinement package

Abstract

Macromolecular crystallographic refinement relies on sometimes dubious stereochemical restraints and rudimentary energy functionals to ensure the correct geometry of the model of the macromolecule and any covalently bound ligand(s). The ligand stereochemical restraint file (CIF) requires a priori understanding of the ligand geometry within the active site, and creation of the CIF is often an error-prone process owing to the great variety of potential ligand chemistry and structure. Stereochemical restraints have been replaced with more robust functionals through the integration of the linear-scaling, semiempirical quantum-mechanics (SE-QM) program DivCon with the PHENIX X-ray refinement engine. The PHENIX/DivCon package has been thoroughly validated on a population of 50 protein-ligand Protein Data Bank (PDB) structures with a range of resolutions and chemistry. The PDB structures used for the validation were originally refined utilizing various refinement packages and were published within the past five years. PHENIX/DivCon does not utilize CIF(s), link restraints and other parameters for refinement and hence it does not make as many a priori assumptions about the model. Across the entire population, the method results in reasonable ligand geometries and low ligand strains, even when the original refinement exhibited difficulties, indicating that PHENIX/DivCon is applicable to both single-structure and high-throughput crystallography.

Keywords: X-ray crystallography; high-throughput crystallography; ligand strain; quantum-mechanics refinement; regional refinement; stereochemical restraints.

Figure 1

Refinement flow-chart in PHENIX.

Figure 2

Schematic view of the region-refinement concept.

Figure 3

Heat of formation (kcal mol⁻¹) computed in DivCon for the structure 1lri after each macro-cycle of the full QM refinement.

Figure 4

The σ_A-weighted 2mF _o − DF _c electron-density map contoured at 1σ around the ligand in the structure 1lri after full (green) and regional (cyan) QM refinement. The density map for other residues in the structure is omitted for clarity.

Figure 5

Superimposition of the QM re-refined PDB structure 2wue (green) with the original PDB structure (yellow).

Figure 6

Superimposition of the QM re-refined PDB structure 2zl9 (green) with the original PDB structure (magenta).

Figure 7

Superimposition of the residues in the coordination sphere of zinc in the structure 2x7t from the regional QM (green) refinements as well as the original PDB structure (magenta).

Figure 8

Superimposition of the QM re-refined PDB structure 3nck (green) with the original PDB structure (yellow).

Figure 9

Superimposition of the QM re-refined PDB structure 3lxs (green) with the original PDB structure (yellow). The difference density is drawn at the 3σ level using the structure factors and phases for the original structure as obtained from the Electron Density Server at Uppsala.