Microtubule nucleation is controlled by the γ-tubulin ring complex (γTuRC) and related γ-tubulin complexes, providing spatial and temporal control over the initiation of microtubule growth. Recent structural work has shed light on the mechanism of γTuRC-based microtubule nucleation, confirming the long-standing hypothesis that it functions as a microtubule template. Crystallographic analysis of the first non-γ-tubulin γTuRC component (GCP4) has resulted in a new appreciation of the relationships among all γTuRC proteins, leading to a refined model of their organization and function. The structures have also suggested an unexpected mechanism for regulating γTuRC activity via conformational modulation of the complex component GCP3. New experiments on γTuRC localization extend these insights, suggesting a direct link between attachment at specific cellular sites and activation.

A crystal structure of human fibrinogen has been determined at approximately 3.3 A resolution. The protein was purified from human blood plasma, first by a cold ethanol precipitation procedure and then by stepwise chromatography on DEAE-cellulose. A product was obtained that was homogeneous on SDS-polyacrylamide gels. Nonetheless, when individual crystals used for X-ray diffraction were examined by SDS gel electrophoresis after data collection, two species of alpha chain were present, indicating that some proteolysis had occurred during the course of operations. Amino-terminal sequencing on post-X-ray crystals showed mostly intact native alpha- and gamma-chain sequences (the native beta chain is blocked). The overall structure differs from that of a native fibrinogen from chicken blood and those reported for a partially proteolyzed bovine fibrinogen in the nature of twist in the coiled-coil regions, likely due to weak forces imparted by unique crystal packing. As such, the structure adds to the inventory of possible conformations that may occur in solution. Other features include a novel interface with an antiparallel arrangement of beta chains and a unique tangential association of coiled coils from neighboring molecules. The carbohydrate groups attached to beta chains are unusually prominent, the full sweep of 11 sugar residues being positioned. As was the case for native chicken fibrinogen, no resolvable electron density could be associated with alphaC domains.

Microtubules are nucleated in vivo by γ-tubulin complexes. The 300 kDa γ-tubulin small complex (γTuSC), consisting of two molecules of γ-tubulin and one copy each of the accessory proteins Spc97p and Spc98p, is the conserved, essential core of the microtubule nucleating machinery1,2. In metazoa multiple γTuSCs assemble with other proteins into γ-tubulin ring complexes (γTuRCs). The structure of γTuRC suggested that it functions as a microtubule template2–5. Because each γTuSC contains two molecules of γ-tubulin, it was assumed that the γTuRC-specific proteins are required to organize γTuSCs to match thirteen-fold microtubule symmetry. Here, we show that γTuSC forms rings even in the absence of other γTuRC components. The yeast adaptor protein Spc110p stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8Å cryo-EM reconstruction of the filament reveals thirteen γ-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97p and Spc98p suggest functions for conserved sequence motifs, with implications for the γTuRC-specific proteins. The γTuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.