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- EMDB-22713: Subtomogram average of flagellar axoneme doublet from the Tetrahy... -

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Basic information

Entry
Database: EMDB / ID: EMD-22713
TitleSubtomogram average of flagellar axoneme doublet from the Tetrahymena thermophila Fap115 knockout mutant
Map data
Sampleflagellum
Biological speciesTetrahymena thermophila (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 17.3 Å
AuthorsFabritius 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
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM127571 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118099 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1S10OD020054 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1S10OD021741 United States
CitationJournal: 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.
History
DepositionSep 25, 2020-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateOct 27, 2021-
Current statusOct 27, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_22713.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.3 Å/pix.
x 140 pix.
= 742. Å
5.3 Å/pix.
x 140 pix.
= 742. Å
5.3 Å/pix.
x 140 pix.
= 742. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.3 Å
Density
Contour LevelBy AUTHOR: 4.07 / Movie #1: 4.07
Minimum - Maximum-19.729034 - 24.488508
Average (Standard dev.)0.010748332 (±1.9044642)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin22138
Dimensions140140140
Spacing140140140
CellA=B=C: 742.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.35.35.3
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z742.000742.000742.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS22138
NC/NR/NS140140140
D min/max/mean-19.72924.4890.011

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Supplemental data

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Sample components

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Entire flagellum

EntireName: flagellum
Details: flagellar axoneme isolated from Tetrahymena thermophila
Number of Components: 1

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Component #1: cellular-component, flagellum

Cellular-componentName: flagellum
Details: flagellar axoneme isolated from Tetrahymena thermophila
Recombinant expression: No
SourceSpecies: Tetrahymena thermophila (eukaryote) / Strain: Fap115 knockout
Source (natural)Organelle: flagellum

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Experimental details

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Sample preparation

SpecimenSpecimen State: Cell / Method: cryo EM
Sample solutionpH: 7
VitrificationCryogen Name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 80 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 33000.0 X (nominal) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image processing

ProcessingMethod: subtomogram averaging / Applied Symmetry: C1 (asymmetric) / Number of Subtomograms: 5926
3D reconstructionAlgorithm: BACK PROJECTION / Software: Xmipp / Resolution: 17.3 Å / Resolution Method: FSC 0.143 CUT-OFF

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