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 rig id-body simulated-annealing refinement of polyalanine models employing succ essively fewer residues per rigid body (down to one alanine residue per bod y) at a very high initial temperature (of the order of T-0 = 10000 K) and w ith the geometric energy terms switched on, not only preserves the geometry of the model but can also converge to an essentially correct polyalanine t race of the target structure, even when the starting model deviates systema tically and significantly from the sought structure. As an example of the a pplication of the method, details are presented of the structure determinat ion of the Ala31Pro mutant of the Rop protein, where an initial roughly pos itioned polyalanine model (giving an average phase difference of 78.2 degre es from the final phase set) was successfully refined against a 1.8 Angstro m resolution native data set, leading to an essentially correct model of th e main chain with an average displacement of its atomic positions from the final model of 0.275 Angstrom The phases calculated from this refined polya lanine 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 i nterpretable electron-density map.