Towards an atomic-resolution structure of the universal microtubule nucleator: the gamma-tubulin ring complex
EMSL Project ID
50666
Abstract
Formation of microtubules (MT) is the basis for segregation of chromosomes during mitosis and establishing the cellular cytoskeleton. MT formation relies on multiple MT nucleation pathways that need to be coordinated in space and time that are catalyzed by the gamma-Tubulin Ring Complex (gamma-TuRC). Despite this, it is unknown how gamma-TuRC facilitates MT nucleation and only low-resolution structures of gamma-TuRC exists limiting our knowledge of its molecular function. In this proposal, we seek to determine the structural basis for gamma-TuRC-mediated MT nucleation by determining the first pseudo atomic-resolution structure of gamma-TuRC using single-particle cryo-electron microscopy (cryo-EM). Currently, we have collected initial data of gamma-TuRC, but data processing has revealed it adopts a preferred orientation during freezing. As such, we have used two approaches to combat this: 1) amphipol A8-35 treatment, where gamma-TuRC samples are treated with 0.1 or 0.01 mg/ml A8-35 prior to freezing, and 2) coupling gamma-TuRC to a MT, where gamma-TuRC can be stabilized in a single conformation, which may aid in observing more orientations of individual particles. Therefore, we are requesting 24 hours of rapid access data collection time to screen 14 grids to reveal if treatment with A8-35 or coupling to a MT, will result in adoption of more observable orientations of gamma-TuRC particles for structure determination. As gamma-TuRC-mediated MT nucleation builds the mitotic spindle body and drives mitosis, determining its structure will be provide novel insights into this process and contribute to discovering cures for diseases that lie at the heart of cell division.
Project Details
Start Date
2019-01-14
End Date
2019-04-10
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members