Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae

Oxysterol-binding protein (OSBP) related proteins (ORPs) are conserved from yeast to humans and are implicated in regulation of sterol homeostasis and in signal transduction pathways. Osh3 of Saccharomyces cerevisiae is a pleckstrin-homology (PH) domain-containing ORP member that regulates phosphoinositide metabolism at endoplasmic reticulum–plasma membrane contact sites. The N-terminal PH domain of Osh3 was purified and crystallized as a lysozyme fusion and the resulting crystal diffracted to 2.3 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 98.03, b = 91.31, c = 84.13 Å, β = 81.41°. With two molecules in the asymmetric unit, the Matthews coefficient was 3.13 ų Da⁻¹. Initial attempts to solve the structure by molecular-replacement techniques using T4 lysozyme as a search model were successful. The C-terminal OSBP-related domain (OBD) of Osh3 was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 1.5 Å resolution. The crystal was orthorhombic, belonging to space group P2₁2₁2₁, with unit-cell parameters a = 41.57, b = 87.52, c = 100.58 Å. With one molecule in the asymmetric unit, the Matthews coefficient was 2.01 ų Da⁻¹. Initial attempts to solve the structure by the single-wavelength anomalous dispersion technique using bromine were successful.