Abstract
Microtubules (MTs) perform essential functions in the cell, and it is critical that they are made at the correct cellular location and cell cycle stage. This nucleation process is catalyzed by the γ-tubulin ring complex (γ-TuRC), a cone-shaped protein complex composed of over 30 subunits. Despite recent insight into the structure of vertebrate γ-TuRC, which shows that its diameter is wider than that of a MT, and that it exhibits little of the symmetry expected for an ideal MT template, the question of how γ-TuRC achieves MT nucleation remains open. Here, we utilized single particle cryo-EM to identify two conformations of γ-TuRC. The helix composed of 14 γ-tubulins at the top of the γ-TuRC cone undergoes substantial deformation, which is predominantly driven by bending of the hinge between the GRIP1 and GRIP2 domains of the γ-tubulin complex proteins. However, surprisingly, this deformation does not remove the inherent asymmetry of γ-TuRC. To further investigate the role of γ-TuRC conformational change, we used cryo electron-tomography (cryo-ET) to obtain a 3D reconstruction of γ-TuRC bound to a nucleated MT, providing insight into the post-nucleation state. Rigid-body fitting of our cryo-EM structures into this reconstruction suggests that the MT lattice is nucleated by spokes 2 through 14 of the γ-tubulin helix, which entails spokes 13 and 14 becoming more structured than what is observed in apo γ-TuRC. Together, our results allow us to propose a model for conformational changes in γ-TuRC and how these may facilitate MT formation in a cell.