A Challenging Case for Protein Crystal Structure Determination: the Mating Pheromone Er-1 from Euplotes raikovi

Four different phasing methods have been applied to the determination of the crystal structure of the 40 amino-acid mating pheromone of the unicellular ciliated protozoan Euplotes raikovi. The difficulties, failures and successes in attempts to solve the structure by: (1) molecular replacement, (2) direct phasing using the `Shake and Bake' algorithm, (3) isomorphous replacement, and (4) multiple-wavelength anomalous disper- sion are described. The structure was first solved by molecular replacement, and then was the first successful structure determination by `Shake and Bake' without the direct involvement of its authors. A description of the current status of the high-resolution refinement of the structure is also given. The model is refined against 1 Å resolution data to an R factor of 12.9%, and includes H atoms and discretely disordered side chains.