Use of single isomorphous replacement data of proteins - resolving the phase ambiguity and a new procedure for phase extension.

Abstract

A procedure combining direct methods and solvent flattening to break the phase ambiguity intrinsic to the single isomorphous replacement (SIR) technique has been tested with the experimental SIR data of the known protein RNase Sa at 2.5 A resolution. The use of direct methods provided better initial phases for the solvent-flattening procedure, while the solvent-flattening procedure greatly improved direct-method phases leading to a traceable Fourier map. A small subset of known phases at low resolution makes direct phasing of SIR data much easier. Accordingly a method for extending low-resolution phases to high-resolution ones is proposed making use of additional SIR information. This reduces the problem of finding a value in the range of 0-2pi for each unknown phase to that of just making a choice between two possible values. Tests with the known protein RNase Sa showed that the method is able to extend phases from a resolution of 6 to 2.5 A leading to an easily traceable Fourier map. The solvent-flattening technique and the combination of which with direct methods were used for the phase extension. Either procedure yielded reasonably good results, but on the whole, the result from the combination of direct methods with solvent flattening is better. Results of the latter procedure were further compared with that from direct phasing of the 2.5 A SIR data and with that from phase extension by solvent flattening without SIR information. An improvement gained by the use of SIR information is evident.