National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM140813
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM127571-05A1
United States
Citation
Journal: J Cell Biol / Year: 2024 Title: Poc1 bridges basal body inner junctions to promote triplet microtubule integrity and connections. Authors: Marisa D Ruehle / Sam Li / David A Agard / Chad G Pearson / Abstract: Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT ...Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. The full localization of the inner scaffold protein Fam161A requires Poc1. As ciliary forces are increased, Fam161A is reduced, indicative of a force-dependent molecular remodeling of the inner scaffold. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces.
Entire : Triplet microtubule from basal body isolated from Tetrahymena the...
Entire
Name: Triplet microtubule from basal body isolated from Tetrahymena thermophila
Components
Organelle or cellular component: Triplet microtubule from basal body isolated from Tetrahymena thermophila
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Supramolecule #1: Triplet microtubule from basal body isolated from Tetrahymena the...
Supramolecule
Name: Triplet microtubule from basal body isolated from Tetrahymena thermophila type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)
Organism: Tetrahymena thermophila (eukaryote)
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Experimental details
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Structure determination
Method
cryo EM
Processing
subtomogram averaging
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.5 / Details: 1xTE
Vitrification
Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK III
Details
Basal bodies are isolated from mutant strain POC1 knockout.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Temperature
Min: 78.0 K / Max: 78.0 K
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number real images: 1 / Average exposure time: 0.36 sec. / Average electron dose: 1.33 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Number classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 25.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) Details: The final average was mirrored after merging two-half maps Number subtomograms used: 868
Extraction
Number tomograms: 83 / Number images used: 10710 / Software - Name: SPIDER
Final 3D classification
Software - Name: RELION
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)
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