USE OF SINGLE ISOMORPHOUS REPLACEMENT DATA OF PROTEINS - RESOLVING THE PHASE AMBIGUITY AND A NEW PROCEDURE FOR PHASE EXTENSION

A procedure combining direct methods and solvent flattening to break t he phase ambiguity intrinsic to the single isomorphous replacement (SI R) technique has been tested with the experimental SIR data of the kno wn protein RNase Sa at 2.5 Angstrom resolution. The use of direct meth ods provided better initial phases for the solvent flattening procedur e, while the solvent-flattening procedure greatly improved direct-meth od 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-reso lution ones is proposed making use of additional SIR information. This reduces the problem of finding a value in the range of 0-2 pi for eac h unknown phase to that of just making a choice between two possible v alues. Tests with the known protein RNase Sa showed that the method is able to extend phases from a resolution of 6 to 2.5 Angstrom leading to an easily traceable Fourier map. The solvent-flattening technique a nd the combination of which with direct methods were used for the phas e extension. Either procedure yielded reasonably good results, but on the whole, the result from the combination of direct methods with solv ent flattening is better. Results of the latter procedure were further compared with that from direct phasing of the 2.5 Angstrom SIR data a nd with that from phase extension by solvent flattening without SIR in formation. An improvement gained by the use of SIR information is evid ent.