Structure determination of human semaphorin 4D as an example of the use of MAD in non-optimal cases

Semaphorins are an important class of signalling molecules involved in axon guidance, immune function and angiogenesis. They are characterized by having an extracellular sema domain of about 500 residues. The steps involved in the determination of the structure of human semaphorin 4D are described here as a case study of selenium MAD phasing in a difficult case with low symmetry, moderate diffraction and low selenium content. A particular feature of this study was the large number of diffraction images required to give data of sufficient quality for structure determination and these data are re-analyzed here to investigate the effects of radiation damage on eventual data quality and to suggest strategies for successful MAD phasing in similar difficult cases.