HAUS augmin like complex subunit 6 L homeolog / HAUS augmin like complex subunit 7 S homeolog / Tubulin alpha-1B chain / HAUS augmin-like complex subunit 8 / Tubulin beta-2B chain Similarity search - Component
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
F32 GM142149
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
K99 GM152794
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01 GM138854
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01 GM141100
United States
Citation
Journal: Nat Commun / Year: 2025 Title: How augmin establishes the angle of the microtubule branch site. Authors: Sophie M Travis / Jodi Kraus / Collin T McManus / Kiana Golden / Rui Zhang / Sabine Petry / Abstract: How microtubules (MTs) are generated in the proper orientation is essential to understanding how the cytoskeleton organizes a cell and MT-dependent events such as cell division. In the spindle, most ...How microtubules (MTs) are generated in the proper orientation is essential to understanding how the cytoskeleton organizes a cell and MT-dependent events such as cell division. In the spindle, most MTs are generated through the branching MT nucleation pathway. In this pathway, new MTs are nucleated from the side of existing MTs and oriented at a shallow angle by the branching factor augmin, ensuring that both MTs have the same polarity. Yet, how augmin binds MTs and sets the branch angle has remained unclear. Here, we report the cryo-electron microscopy structure of an augmin subcomplex on the MT. This structure resembles that of NDC80 bound to the MT, with the conserved CH domain of augmin's Haus6 subunit directly proximal to the MT lattice. We find that the Haus6 CH domain is a bona fide MT binding site that increases augmin's affinity for the MT and helps establish branch angle. A second binding site, located in the disordered N-terminus of Haus8, also establishes branch angle,. Thus, we find that augmin regulates MT branching using two domains, each tuned to modulate MT affinity and MT branch angle. This work expands our mechanistic understanding of branching MT nucleation and thus spindle formation.
Model: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.037 kPa
Vitrification
Cryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
Details
20 uM tubulin, 10 uM augmin T-II bonsai
-
Electron microscopy
Microscope
TFS KRIOS
Specialist optics
Energy filter - Name: TFS Selectris / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 5012 / Average exposure time: 4.1 sec. / Average electron dose: 20.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Type of model: OTHER Details: Both 13-protofilament and 14-protofilament reference models were given, as detailed in Valdez et al 2023 DOI: 10.1101/2023.12.18.571906
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 99011
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