Journal: J Cell Sci / Year: 2020 Title: The structure and symmetry of the radial spoke protein complex in flagella. Authors: Emiliya Poghosyan / Ioan Iacovache / Lenka Faltova / Alexander Leitner / Pinfen Yang / Dennis R Diener / Ruedi Aebersold / Benoit Zuber / Takashi Ishikawa / Abstract: The radial spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The radial spoke ...The radial spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The radial spoke has three regions - a spoke head, a bifurcated neck and a stalk. Although the neck and the stalk are asymmetric, twofold symmetry of the head has remained controversial. In this work we used single particle cryo-electron microscopy (cryo-EM) analysis to generate a 3D structure of the whole radial spoke at unprecedented resolution. We show the head region at 15 Å (1.5 nm) resolution and confirm twofold symmetry. Using distance constraints generated by cross-linking mass spectrometry, we locate two components, RSP2 and RSP4, at the head and neck regions. Our biophysical analysis of isolated RSP4, RSP9, and RSP10 affirmed their oligomeric state. Our results enable us to redefine the boundaries of the regions and propose a model of organization of the radial spoke component proteins.
History
Deposition
May 27, 2020
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Header (metadata) release
Aug 5, 2020
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Map release
Aug 5, 2020
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Update
Sep 30, 2020
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Current status
Sep 30, 2020
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 7.5 Details: 10 mM HEPES pH 7.5, 5 mM MgSO4, 1 mM BME, 0.5 mM EDTA, 25 mM KCl
Grid
Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 20 K / Instrument: FEI VITROBOT MARK III
Details
Solution of protein complexes isolated from natural source
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Electron microscopy
Microscope
FEI TECNAI F20
Image recording
Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 736 / Average exposure time: 7.0 sec. / Average electron dose: 20.0 e/Å2 / Details: 7 frames/sec
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stage
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 240000
CTF correction
Software - Name: CTFFIND
Startup model
Type of model: OTHER / Details: subtomogram averaging
Final reconstruction
Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 12300
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