Section 19.1.7. D₂O − H₂O solvent difference maps

D₂O − H₂O solvent difference maps provide an unbiased method for identifying water molecules and exchangeable hydrogens (Kossiakoff et al., 1992). For several years, the large difference in the scattering characteristics of neutrons by H₂O compared to D₂O has been effectively exploited by using density matching and exchange labelling in small-angle neutron-scattering experiments. This difference can likewise be exploited in neutron protein crystallography to determine the detailed structural characteristics of protein hydration through the calculation of solvent difference maps (Shpungin & Kossiakoff, 1986; Kossiakoff et al., 1992). In practice, such maps are obtained by comparing the changes in diffracted intensities between two sets of data – one obtained from a crystal having H₂O as the major solvent constituent, and a second where D₂O is the solvent medium. To a good approximation, the protein-atom contributions to the scattering intensities in both data sets are equal and cancel, but since H₂O and D₂O have very different scattering properties, their differences are accentuated to reveal an accurate and nearly unbiased representation of the solvent structure.

The features of a solvent difference map of this type are not as affected by errors in the phasing model as conventional difference Fourier maps. In addition, there are refinement procedures that can be applied to them that lead to significant enhancement in signal/noise discrimination. The basic feature of the method is a set of density-modification steps based on the fact that a considerable amount of information about the density distribution of the crystallographic unit cell is known. For instance, it is known that the region of the unit cell occupied by protein atoms should be featureless in solvent maps. It can also be assumed that, as an approximation, solvent regions further than 4 Å from the protein surface have bulk solvent characteristics and can be treated as a constant density region. Combining these two regions gives about 50–60% of the total volume of the unit cell.

Knowledge of the density content of such a large percentage of the unit cell places a strong constraint on the overall character of the Fourier transform, a fact that can be used to improve the quality of the experimentally determined phases