10779/crick.12482189.v1 Tanja Consolati Tanja Consolati Julia Locke Julia Locke Johanna Roostalu Johanna Roostalu Zhuo Angel Chen Zhuo Angel Chen Julian Gannon Julian Gannon Jayant Asthana Jayant Asthana Wei Ming Lim Wei Ming Lim Fabrizio Martino Fabrizio Martino Milos A Cvetkovic Milos A Cvetkovic Juri Rappsilber Juri Rappsilber Alessandro Costa Alessandro Costa Thomas Surrey Thomas Surrey Microtubule nucleation properties of single human γTuRCs explained by their cryo-EM structure. The Francis Crick Institute 2020 CLMS MZT2 TIRF microscopy TPX2 actin chTOG cryo-electron microscopy microtubule nucleation γ-tubulin ring complex γTuRC structure Costa FC001065 SB-ack EM-ack CS-ack FC-ack PRT-ack Surrey FC001163 Developmental Biology 06 Biological Sciences 11 Medical and Health Sciences 2020-06-25 14:11:58 Journal contribution https://crick.figshare.com/articles/journal_contribution/Microtubule_nucleation_properties_of_single_human_TuRCs_explained_by_their_cryo-EM_structure_/12482189 The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.