All model refinement was performed using the automated refinement procedure(ARP) developed by lamzin and Wilson. The procedure combines the least square refinement technique with the ‘BAD ATOM’ rejection criteria. The program automatically detects where new atoms should be and places them there. Since the complete lysozymes model was already available the procedure was principally used to locate and refine the positions and B-factors of the solvent water molecules. Although ARP may be applied to the refinement of incomplete protein models it will be discussed here only in terms of its use in adding ordered solvent.
ARP has three main steps:
1. restrained least squares refinementa against the observed X-ray data.
2. atom rejection and inclusion on the basis of electron density maps calculated using the coefficients.
3. real space refinement of all the solvent molecules.
Least squares refinement using X-ray data with maximum resolution of 2.0A or worse must generally be accompanied by the application of stereo chemical restraints since at these lower resolution the number of parameters to be refined can sometimes exceed the number of observables. The inclusion of restraints in the refinement complements the X-ray data by effectively increasing the number of observables. The three programes used in the least squar minimization stages are were: SFALL,PROTIN,PROLSQ. The restraints take the form of a library of stereo chemical data which contains a collection of target bond length and bond angles established by small molecules studies of 20amino acids and high resolute studies of small peptide structure and proteins. The function F to the minimized in restrained I.S refinement is made up of a diffraction term,plus restraining terms for bond distances,planarity of the atoms in the ring molecules such as histidine and phenyalanine,close contact restraints for non bonded atoms, chirality and bonded angle restraints and restraints on the isotropic Debye-waller factors of B-factors.
The calculations and automated evaluation of the electron density maps using the calculated phases from the model form the basis of the ARP procedure. The maps are used to judge whether solvent molecules should be removed from of included into the model. Solvent molecules in the model which sit in weak density are good candidates for the removal due to the density coming from the observed structure factors. During each ARP cycle a maximum of 4 solvent molecules were removed in the way. Similarly a maximum of 4 molecules were included at positions corresponding to large positive density in different maps. Additional criteria such as water peak sphericity and nearest neighbour distances are used as measure of the integrity of the solvent structures..
Finally in the ARP cycle real space refinement is applied to the water molecules.