Water occupies typically 50% of a protein crystal and thus significantly contributes to the diffraction signal in crystallography experiments. Separating its contribution from that of the protein is however challenging because most water molecules are not localized and are thus difficult to assign to specific density peaks. The intricateness of the protein-water interface compounds this difficulty. This information has therefore not often been used to study biomolecular solvation. Here we develop a methodology to surmount in part this difficulty. More specifically we compare the solvent structure obtained from diffraction data for which experimental phasing is available to that obtained from constrained molecular dynamics (MD) simulations. The resulting spatial density maps show that commonly used MD water models are only partially successful at reproducing the structural features of biomolecular solvation. The radial distribution of water is captured with only slightly higher accuracy than its angular distribution and only a fraction of the water molecules assigned with high reliability to the crystal structure are recovered. These differences are likely due to shortcomings of both the water models and the protein force fields. Despite these limitations we achieve to infer protonation states of some of the side chains utilizing MD-derived densities.
