Solvent structure in crystals of trypsin determined by X‐ray and neutron diffraction

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X‐ray diffraction to 1.35 Å resolution and by neutron diffraction to 2.1 Å resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X‐ray diffraction, which were verified by comparison to D2OH2O difference neutron scattering density. Six of 184 water molecules in the X‐ray structure, all with B‐factors greater than 50 Å2, were found to be spurious after comparison with neutron results. Roughly two‐thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one‐half of the water of hydration was located by X‐ray diffraction. Polar regions of the protein are well hydrated, and significant D2OH2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures.