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Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.

Developmental cell (2020-05-21)
Tanja Consolati, Julia Locke, Johanna Roostalu, Zhuo Angel Chen, Julian Gannon, Jayant Asthana, Wei Ming Lim, Fabrizio Martino, Milos A Cvetkovic, Juri Rappsilber, Alessandro Costa, Thomas Surrey
ABSTRACT

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.

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