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Figure 4
Visualization of the 2mFo − DFc and mFo − DFc maps covering residues 298–300 in β-lactamase (PDB entry 6mu9; Center for Structural Genomics of Infectious Diseases, unpublished work). The 2mFo − DFc map (blue mesh) represents the electron density corresponding to the refined structure, while the mFo − DFc map (green mesh for positive difference density) highlights regions where significant deviations are observed between the model and the experimental data. Carbon, nitrogen and oxygen atoms are shown in green, blue and red, respectively. Hydrogen atoms, where present, are depicted in white. Figures were prepared using PyMOL (Schrödinger). (a) The structure and maps correspond to the original REFMAC5 refinement deposited in the PDB. A difference density peak near the carbonyl oxygen atom (O-299) is observed at a distance of 1.03 Å, with a ∠C–O–peak centre angle of 92.5°. Additional difference densities are observed for hydrogen atoms at the Cα, Cβ and N atoms of residue 299, as well as at the carbonyl oxygen atom and Cα atoms of residue 298. The maps are contoured at 1.8 e Å−3 (2mFo − DFc) and 0.37 e Å−3 (mFo − DFc). (b) The structure refined in Phenix without hydrogen atoms shows a similar difference density pattern to that in (a), particularly near the carbonyl oxygen atom (O-299). The maps are contoured at 2.8 e Å−3 (2mFo − DFc) and 0.37 e Å−3 (mFo − DFc). (c) The structure refined in Phenix with hydrogen atoms in riding positions shows stronger difference density near the carbonyl oxygen atom of residue 299, with weaker density observed for the carbonyl oxygen atom of residue 298. This indicates an improved fit of the model to the experimental data when hydrogen atoms are included. (d) The structure refined in Phenix with hydrogen atoms in riding positions and a protonated carbonyl oxygen atom at residue 299 demonstrates further refinement accuracy. The hydrogen atom attached to the protonated carbonyl oxygen atom is well refined, with no residual difference density at this site, indicating correct placement. Additionally, the hydrogen atom from the riding position of the N atom at residue 299 was removed for validation purposes. Stronger difference density is observed near the N atom of residue 299, while weaker density is seen near the carbonyl oxygen of residue 298. Across all figures, no difference density is observed at the N atom of residue 300, validating its structural integrity. These observations highlight the importance of accurately modelling hydrogen placement and protonation states to improve structural refinement quality.

IUCrJ
Volume 12| Part 3| May 2025|
ISSN: 2052-2525
https://doi.org/10.1107/S2052252525002106