The centrosome plays a fundamental role in organizing the microtubule (MT) cytoskeleton. Defects in centrosome function lead to errors in chromosome segregation during cell division and play a major role in both the initiation of carcinogenesis and in the progression of cancer. The studies outlined in this proposal will seek to understand the mechanisms of microtubule nucleation by focusing on the structure of a key hetero-tetrameric molecular complex that underlies MT nucleation in all eukaryotes. A combination of EM single particle reconstruction methods and X-ray crystallography is proposed to determine the 3D structure of the yeast tub4 and/or the drosophila gamma-tubulin small complex. The goal of the EM work will be to determine the overall organization of the tub4 complex and discover which gamma-tubulin surfaces are exposed, and to map the location where spd 10 binds. High resolution crystallographic studies will provide unprecedented information on the structure of the complex, the conformation of gamma-tubulin, and the role of nucleotide. Understanding the mechanism, structure, composition, and regulation of microtubule nucleation sites in the centrosome will provide significant insight into how centrosome defects can lead to cancer.