|Entry||Database: EMDB / ID: EMD-22713|
|Title||Subtomogram average of flagellar axoneme doublet from the Tetrahymena thermophila Fap115 knockout mutant|
|Biological species||Tetrahymena thermophila (eukaryote)|
|Method||subtomogram averaging / cryo EM / Resolution: 17.3 Å|
|Authors||Fabritius AS / Bayless BA / Li S / Stoddard D / Heydeck W / Ebmeier CC / Anderson L / Gunnels T / Nachiappan C / Whitall J ...Fabritius AS / Bayless BA / Li S / Stoddard D / Heydeck W / Ebmeier CC / Anderson L / Gunnels T / Nachiappan C / Whitall J / Old W / Agard DA / Nicastro D / Winey M|
|Funding support|| United States, 4 items |
|Citation||Journal: Mol Biol Cell / Year: 2021|
Title: Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null cells reveals functional MIPs.
Authors: Amy S Fabritius / Brian A Bayless / Sam Li / Daniel Stoddard / Westley Heydeck / Christopher C Ebmeier / Lauren Anderson / Tess Gunnels / Chidambaram Nachiappan / Justen B Whittall / William ...Authors: Amy S Fabritius / Brian A Bayless / Sam Li / Daniel Stoddard / Westley Heydeck / Christopher C Ebmeier / Lauren Anderson / Tess Gunnels / Chidambaram Nachiappan / Justen B Whittall / William Old / David A Agard / Daniela Nicastro / Mark Winey /
Abstract: The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of ...The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of microtubule inner proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist . Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in knockout axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_22713.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 5.3 Å|
|Symmetry||Space group: 1|
CCP4 map header:
Details: flagellar axoneme isolated from Tetrahymena thermophila
Number of Components: 1
-Component #1: cellular-component, flagellum
Details: flagellar axoneme isolated from Tetrahymena thermophila
Recombinant expression: No
|Source||Species: Tetrahymena thermophila (eukaryote) / Strain: Fap115 knockout|
|Source (natural)||Organelle: flagellum|
|Specimen||Specimen State: Cell / Method: cryo EM|
|Sample solution||pH: 7|
|Vitrification||Cryogen Name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 80 e/Å2 / Illumination Mode: FLOOD BEAM|
|Lens||Magnification: 33000.0 X (nominal) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Processing||Method: subtomogram averaging / Applied Symmetry: C1 (asymmetric) / Number of Subtomograms: 5926|
|3D reconstruction||Algorithm: BACK PROJECTION / Software: Xmipp / Resolution: 17.3 Å / Resolution Method: FSC 0.143 CUT-OFF|
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