With the aim of determining the actual positions and occupancies of the Iridium atoms in the lysozyme via difference Fourier techniques native data collected to 2.25Å resolution were used to refine the lysozyme structure further and to include ordered solvent into the model so as to obtain a more accurate set of calculated phases 's.
The method used for the inclusion of extra ordered solvent molecules was the Automated Refinement Procedure (ARP) which is described in Sec. .
The initial model with no added water molecules had an R-factor of for all reflections. The refinement proceeded mostly automatically over 140 cycles of least-squares minimisation, solvent modification and solvent refinement. It was necessary towards the end of the refinement to correct several residues of the model by hand. One of these was Glu 35 whose conformation, as expected, differed slightly from that in the model of hen (Gallus Gallus) egg-white iodine inactivated lysozyme  obtained from the Protein Data Bank .
A total of 116 water molecules were added to the model and resulted in a final R-factor of for all reflections and for all reflections with . On the whole the model refined satisfactorily and on inspection of the electron density map all except 5 residues in the model had all atoms within density greater than Rms density. The five poorly defined residues were Arg 73, Asp 119, Gln 121, Arg 125, Leu 129, all of which are on the surface of the protein molecule extending into the solvent region and therefore prone to conformational disorder. Leu 129 is the C terminal residue of the structure. All of the water molecules except five had B-factors less than 70Å and during the refinement three of the waters were assigned half occupancies because of their special positions on two fold axes. Fig. shows a cross section through the electron density map along with the refined protein model for native lysozyme.
Figure: Cross section through the electron density map calculated with coefficients and phases from the refined native model (also shown). The stick model is coloured according to atomic species (yellow:carbon, blue:nitrogen, red:oxygen, green:sulphur. ARP water sites are shown as red crosses). The map is contoured at Rms density. Visible in the upper left quadrant of the diagram is the loop region extending from Tyr 53 to Trp 62. Two of the water molecules found during ARP refinement are also shown. Wat 10 was refined with a B-factor of 11.2Å while Wat 95 had a B-factor of 53.1Å. (The diagram was produced using O, ODLEDIT and OPLOT.)