PROTEIN CRYSTAL SEEDING STUDIED BY PHASE DIAGRAMS. Qing Han and Sheng-Xiang Lin, MRC Group in Molecular Endocrinology, CHUL Research Center and Laval University, Québec, G1V 4G2, Canada

Though different seeding techniques of macromolecular crystallization were explored and successful examples were reported, the seeding technique is not yet a standard method in macromolecule crystallization. The main reason is that we lack basic knowledge about macromolecule seeding process. In this report the seeding phase diagrams were determined by the batch experiments using hen egg lysozyme and human 17ß-hydroxysteroid dehydrogenase. On the basis of the growth of microcrystals, the phase diagram could be divided into five areas: damage region, equilibration region, unobvious growth region, obvious growth region, nucleation region. The ideal seeding, in which the microcrystal can grow evidently without dissolution and new nucleation, was only achieved in a narrow domain of phase diagram. The solution of high protein concentration with low salt concentration is shown to be beneficial for seeding. The broken mincrocrystal and aggregation that were introduced to new drops could cause nucleation at low level of supersaturation. The techniques of microcrystal wash, crystal transfer and crystal soaking are also described. These results will be useful for rational seeding in macromolecular crystallization.