Section 25.2.5.2.  ARP/wARP applications

The hybrid models described above are used as the main tool for obtaining as full a protein model as possible from the map calculated with the initial phases.

Given the information contained in the hybrid model in the traditional form of stereochemical restraints, reciprocal-space refinement can work more efficiently, new improved phases can be obtained and a more accurate and complete protein model can be constructed. The new hybrid model can be re-input to refinement and these steps can be iterated so that improved phases result in construction of ever larger parts of the protein. An almost complete protein model can be obtained in a fully automated way.

Starting from a molecular-replacement solution implies that a search model positioned in the new lattice is already available. The model can be directly incorporated in restrained ARP refinement. If the starting model is very incomplete or different, its atoms can be regarded as free atoms and the solution can be treated as starting from just initial phases. This increases the radius of convergence and minimizes the bias introduced by the search model.

Slightly varying the protocol described for generating models from maps results in a set of slightly different free-atom models. Each model is then submitted to ARP. In protein crystallography, there are generally insufficient data for convergence of free-atom refinement to a global minimum and different starting models result in final models with small differences, i.e. containing different errors. Averaging of these models can be utilized to minimize the overall error. The procedure in effect imposes a random noise, small enough to be eliminated during the subsequent averaging, but large enough to overcome at least some of the systematic errors.

Structure factors are calculated for all the refined models and a vector average of the calculated structure factors is derived. The phase of the vector average is more accurate than that from any of the individual models. A weight, W_wARP, is assigned to each structure factor on the basis of the variance of the two-dimensional distribution of the individual structure factors around the mean. The mean value of W_wARP over all reflections and the R factor after averaging can be used to judge the progress of the averaging procedure.

If the coordinates of one or a few heavy atoms are known, initial phases can be calculated. The problem of solving the structure of such a metalloprotein from the sites of the metal alone can be considered in the same framework as for heavy-atom-replacement solutions. Maps calculated from the phases of heavy atoms alone often have the best defined features within a defined radius of the heavy atom(s). Thus protocols that do not place all atoms at the start but instead perform a slow building while extending the model in a growing sphere around the heavy atom are preferred. When such a model is essentially complete, it can be used for automated tracing and completion of the model.

In this application, the protein (or nucleic acid) model is not rebuilt during refinement, and only the solvent structure is continuously updated, allowing the construction of a solvent model without iterative manual map inspection.