Abstract OR-9: Cryo-EM Structure of the Reconstituted Human γ-Tubulin Ring Complex

Background: Microtubules (MTs) are essential cytoskeletal polymers that provide structural support for the cell and play important roles in cell division, motility, and intracellular transport. The γ-tubulin ring complex (γTuRC) is the major MT nucleator in animal cells. The molecular mechanism by which the γTuRC promotes MT nucleation remains poorly understood although a template-based mechanism, remains the most widely accepted (Moritz et al., 2000, Kollman et al., 2010). According to this model γTuRC, a 2 MDa multi-subunit protein complex, forms a lock washer-like structure, in which γ-tubulin molecules are arranged in a ring-shaped structure that serves as a template for the assembly of αβ-tubulin heterodimers. Methods: We have set up an in vitro system to purify the human γTuRC using infected insect cells with recombinant baculoviruses. This complex sample was subjected to cryo-EM analysis and single-particle reconstruction. Results: We have demonstrated that RUVBL1-RUVBL2 AAA-ATPase complex (RUVBL) controls the assembly and composition of γTuRC in human cells both in vivo and in vitro. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous co-expression system. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC (Consolati et al., 2020, Liu et al., 2020, Wieczorek et al., 2020), as revealed by its cryo-EM structure at ~4.0 Å resolution. Conclusion: We have been able to identify novel mechanistic and structural features that determine the intricate, higher-order γTuRC architecture (Zimmermann, Serna et al., 2020)