The ordered solvent was involved in hydrogen bonding to protein and carbohydrate residues (7 water molecules) and in forming more extended hydrogen bonding networks (6 water molecules), which led to greater complementarity between Ley and the hu3S193 binding site [26].
In the vicinity of the Fab1-Ley interaction, two ordered solvent molecules were observed in the 2.5 Å resolution electron density maps (Fig. 2 A) and both occupied the same locations as in the 1S3K structure (Fig. 2 C).
The structure revealed the presence of four ordered solvent water molecules in the apo structure.
In wild-type OYE 2.6, an ordered solvent molecule (WAT 611) is hydrogen-bonded to the side chain of Tyr78.
We sought to determine whether this excess could be attributed to expulsion of residual ordered solvent water molecules from the protein binding pocket.
However, we note that many distinct transitions likely occur across this temperature range as different parts of the bulk solvent and different ordered solvent regions become much more strongly ordered.
For example, XRD analysis of a clathrate often locates ordered solvent molecules within its structure that will often increase the density of the crystal to over 0.9 cm−3, even though the evacuated crystal may be of much lower density.
Our original crystal structure of rMUP in the absence of ligand[ 10] (PDB ID: 1QY0) was not suitable for the observation of ordered solvent water molecules within the binding pocket due to the presence of cryoprotectant glycerol.
No ordered solvent molecule corresponding to WAT 611 was observed in the Tyr78Trp variant, and it would be impossible to place a water at the same position in the mutant because the closest approach distance would be only 1.8 Å.
Subsequent model refinement and addition of ordered solvent was carried out using the autobuild and refinement modules of Phenix with manual rebuilding in Coot, which was also used to introduce the sequence changes into the model (T172R, G173Q, L196C, and I301C).
