Meaningful refinement of polyalanine models using rigid-body simulated annealing: application to the structure determination of the A31P Rop mutant.

Conventional refinement methods, when applied to even correctly positioned polyalanine models of a target structure, result in a systematic distortion of the molecular geometry and to a concomitant increase in the mean phase difference from the correct phase set. Here, it is shown that iterative rigid-body simulated-annealing refinement of polyalanine models employing successively fewer residues per rigid body (down to one alanine residue per body) at a very high initial temperature (of the order of T0 = 10000 K) and with the geometric energy terms switched on, not only preserves the geometry of the model but can also converge to an essentially correct polyalanine trace of the target structure, even when the starting model deviates systematically and significantly from the sought structure. As an example of the application of the method, details are presented of the structure determination of the Ala31Pro mutant of the Rop protein, where an initial roughly positioned polyalanine model (giving an average phase difference of 78.2 degrees from the final phase set) was successfully refined against a 1.8 A resolution native data set, leading to an essentially correct model of the main chain with an average displacement of its atomic positions from the final model of 0.275 A. The phases calculated from this refined polyalanine model had an average difference of 43.8 degrees from the final phase set (corresponding to a mean figure of merit of 0.72) and gave a readily interpretable electron-density map.