Crystallization and preliminary X-ray characterization of the Skp1–Fbg3 complex

The crystallization and preliminary X-ray diffraction studies of the Skp1–Fbg3 complex are reported. Crystallization by repeated microseeding using selected crystals as a source of microseeds is described. F-box proteins are the substrate-recognition components of Skp1–Cullin1–F-box protein–Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 34.1, b = 76.6, c = 193.9 Å and one molecule per asymmetric unit