Crystallization and preliminary X-ray analysis of FlhD from Escherichia coli

The flhD gene from Escherichia coli codes for one of the transcriptional ac tivators of flagellar genes and is a putative global regulator in the cell. FlhD together with FlhC forms a complex that positively regulates the expr ession of flagellar genes. However, FlhD is able to change ifs DNA binding specificity, probably by forming another complex with some unknown factor o r by binding to other targets by itself. FlhD is a good model to study the DNA binding specificity by complex formation. The FlhD three-dimensional st ructure will certainly improve our understanding of this protein, which is the first case of combinatory specificity in prokaryotes. The flhD gene was over-expressed, and the resulting protein was purified to homogeneity. Flh D crystals were obtained from 15-25% PEG 5000, 0.05-0.1 M lithium sulfate, 0.1 M Tris, pH 8.5, or 20-30% PEG 5000, 0.05-0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5, as precipitant solutions, and belong to the space group I4 with unit cell parameters of a = b = 90 Angstrom, c = 42.6 Angstrom and alpha = beta = gamma = 90 degrees and contain two molecules per asymmetric unit. A complete native data set has been collected to 2.3-Angstrom resolu tion. (C) 1998 Academic Press.