Solution of the structure of the cofactor-binding fragment of CysB: A struggle against non-isomorphism

The elucidation of the structure of CysB(88-324) by multiple isomorphous re placement (MIR) techniques was seriously delayed by problems encountered at every stage of the analysis. There was extensive non-isomorphism both betw een different native crystals and between native and heavy-atom-soaked crys tals. The heavy-atom substitution was invariably weak and different soaking experiments frequently led to substitution at common sites. These correlat ed heavy-atom binding sites resulted in an overestimation of the phase info rmation. Missing low-resolution reflections in the native data set, constit uting only 2% of the total observations, reduced the power of density modif ication and phase refinement. Finally, the extensive dimer interface made i t difficult to isolate a single molecule in the course of model building in to the MIR maps. The power of maximum-likelihood refinement (REFMAC) was ex ploited in solving the structure by means of iterative cycles of refinement of a partial model, initially comprising only 30% of the protein atoms in the final coordinate set. This technique, which uses experimental phases, c an automatically discriminate the correct and incorrect parts of electron-d ensity maps and give properly weighted combined phases which are better tha n the experimental or calculated ones. This allowed the model to be gradual ly extended by manual building into improved electron-density maps. A model generated in this way, containing just 50% of the protein atoms, proved go od enough to find the transformations needed for multi-crystal averaging be tween different crystal forms. The averaging regime improved the phasing dr amatically such that the complete model could be built. The problems, final solutions and some possible causes for the observed lack of isomorphism ar e discussed.