Protonation-state determination in proteins using high-resolution X-ray crystallography: Effects of resolution and completeness

A bond-distance analysis has been undertaken to determine the protonation states of ionizable amino acids in trypsin, subtilisin and lysozyme. The diffraction resolutions were 1.2 A ̊ for trypsin (97% complete, 12% H-atom visibility at 2.5 ), 1.26 A ̊ for subtilisin (100% complete, 11% H-atom visibility at 2.5 ) and 0.65 A ̊ for lysozyme (PDB entry 2vb1; 98% complete, 30% H-atom visibility at 3 ). These studies provide a wide diffraction resolution range for assessment. The bond- length e.s.d.s obtained are as small as 0.008 A ̊ and thus provide an exceptional opportunity for bond-length analyses. The results indicate that useful information can be obtained from diffraction data at around 1.2–1.3 A ̊ resolution and that minor increases in resolution can have significant effects on reducing the associated bond-length standard deviations. The protona- tion states in histidine residues were also considered; however, owing to the smaller differences between the protonated and deprotonated forms it is much more difficult to infer the protonation states of these residues. Not even the 0.65 A ̊ resolution lysozyme structure provided the necessary accuracy to determine the protonation states of histidine.