Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod-Zwilch-ZW10 (RZZ) complex

摘要

The spindle-assembly checkpoint (SAC) monitors kinetochore-microtubule attachment during mitosis. In metazoans, the three-subunit Rod-Zwilch-ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1-Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of similar to 800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P3(1) (No. 144) or P3(2) (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 angstrom, alpha = beta = 90.0, gamma = 120.0 degrees.