Section 18.4.5.4. Ordered solvent water

A protein crystal typically contains some 50% aqueous solvent. This is roughly divided into two separate zones. The first is a set of highly ordered sites close to the surface of the protein. The second, lying remote from the protein surface, is essentially composed of fluid water, with no order between different unit cells.

At room temperature, the solvent sites around the surface are assumed to be in dynamic equilibrium with the surrounding fluid, as for a protein in solution. Nevertheless, the observation of apparently ordered solvent sites on the surface indicates that these are occupied most of the time. The waters are organized in hydrogen-bonded networks, both to the protein and with one another. The most ordered water sites lie in the first solvent shell, where at least one contact is made directly to the protein. For the second and subsequent shells, the degree of order diminishes: such shells form an intermediate grey level between the ordered protein and the totally disordered fluid. Indeed, the flexible residues on the surface form part of the continuum between a solid and liquid phase.

In the ordered region, the solvent structure can be modelled by discrete sites whose positional parameters and ADPs can be refined. For sites with low ADPs, the refinement is stable and their behaviour well defined. As the ADPs increase, or more likely the associated occupancy in a particular site falls, the behaviour deteriorates, until finally the existence of the site becomes dubious. There is no hard cutoff for the reality of a weak solvent site. However, the number and significance of solvent sites are increased by atomic resolution data. Despite the fact that the waters contribute only weakly to the high-resolution terms, the improved accuracy of the rest of the structure means that their positions become better defined.

Indeed, the occupancy of some solvent sites can be refined if the resolution is sufficient, or at least their fractional occupancy can be estimated and kept fixed (Walsh et al., 1998). This leads to the possibility of defining overlapping water networks with alternative hydrogen-bonding schemes. This can be a most time consuming step in atomic resolution refinement, and a trade-off finally has to be made between the relevance of any improvement in the model and the time spent.