Detection of alternative conformations by unrestrained refinement

Unrestrained refinement is stable for the vast majority of atoms when working at atomic resolution. Nevertheless, geometrical restraints should be retained in refinement for residues that are present in several (alternative) conformations in the crystal used for the X-ray experiment; otherwise, such residues deteriorate significantly. The authors believe that a large distortion of a residue in unrestrained refinement may hint at the presence of alternative conformations of this residue. To obtain these hints in a routine way, two methods of analyzing the shifts of atomic centres resulting from several cycles of unrestrained refinement are described. A simple diagram plotting the values of the atomic shifts against the residue number may give an idea of the crystallographic order of different parts of the structure at a qualitative level. To put the analysis on a more quantitative basis, several decision-making procedures were developed and tested which compose a list of residues that are likely to be present in alternative conformations or to be disordered and so should be checked thoroughly using Fourier syntheses and included in the model with alternative conformations when necessary. The parameters and performance of the suggested procedures were estimated by the use of 203 PDB structures refined at resolutions better than 1.2 Å. Decision-making procedures based on analysis of atomic shifts were found to be more reliable than similar procedures based on atomic displacement parameters or density values calculated at atomic centres.