RESUMO
The preparation of derivatives by the traditional methods of soaking is one of the most time-consuming steps in protein crystal structure solution by X-ray diffraction techniques. The 'quick cryosoaking' procedure for derivatization with halides (monovalent anions) offers the possibility of significantly speeding up this process [Dauter et al. (2000), Acta Cryst. D56, 232-237]. In the present work, an extension of this technique is proposed and the use of two different classes of compounds (monovalent and polyvalent cations) that can be successfully utilized in the quick cryosoaking procedure for the derivatization and phasing of protein crystals is described. This approach has been tested on hen egg-white lysozyme and has been successfully used to solve the structure of a novel trypsin inhibitor. The possibility of using cations in the fast cryosoaking procedure gives additional flexibility in the process of derivatization and increases the chances of success in phase determination. This method can be applied to high-throughput crystallographic projects.
Assuntos
Proteínas/química , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
Hexameric glucosamine-6-phosphate deaminase from Escherichia coli has been crystallized isomorphously with both phosphate and ammonium sulphate as precipitants, over a wide pH range (6.0 to 9.0). The crystals belong to space group R32 and the cell parameters in the hexagonal setting are a = b = 125.9 A and c = 223.2 A. A complete native data set was collected to 2.1 A resolution. Self-rotation function studies suggest that the hexamers sit on the 3-fold axis and have point group symmetry 32, with a non-crystallographic dyad relating two monomers linked by an interchain disulfide bridge. A possible packing for the unit cell is proposed.