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- EMDB-9766: Doublet microtubule of fap45 null mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-9766
TitleDoublet microtubule of fap45 null mutant
Map datafap45_DMT
Sample
  • Organelle or cellular component: axoneme of Chlamydomonas reinhardtii
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 39.6 Å
AuthorsOwa M / Uchihashi T / Yanagisawa H / Yamano T / Iguchi H / Fukuzawa H / Wakabayashi K / Ando T / Kikkawa M
CitationJournal: Nat Commun / Year: 2019
Title: Inner lumen proteins stabilize doublet microtubules in cilia and flagella.
Authors: Mikito Owa / Takayuki Uchihashi / Haru-Aki Yanagisawa / Takashi Yamano / Hiro Iguchi / Hideya Fukuzawa / Ken-Ichi Wakabayashi / Toshio Ando / Masahide Kikkawa /
Abstract: Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains ...Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.
History
DepositionDec 28, 2018-
Header (metadata) releaseJan 30, 2019-
Map releaseJan 30, 2019-
UpdateMar 20, 2019-
Current statusMar 20, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 60
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 60
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9766.png

Downloads & links

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Map

FileDownload / File: emd_9766.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfap45_DMT
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.1 Å/pix.
x 200 pix.
= 1420. Å		7.1 Å/pix.
x 200 pix.
= 1420. Å		7.1 Å/pix.
x 200 pix.
= 1420. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 60.0 / Movie #1: 60
Minimum - Maximum-384.406830000000014 - 388.135620000000017
Average (Standard dev.)-9.983328 (±48.821674000000002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 1420.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.17.17.1
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z1420.0001420.0001420.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-384.407388.136-9.983

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Supplemental data

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Sample components

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Entire : axoneme of Chlamydomonas reinhardtii

EntireName: axoneme of Chlamydomonas reinhardtii
Components
  • Organelle or cellular component: axoneme of Chlamydomonas reinhardtii

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Supramolecule #1: axoneme of Chlamydomonas reinhardtii

SupramoleculeName: axoneme of Chlamydomonas reinhardtii / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: fap45

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL 3100FFC
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 100.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: DIFFRACTION

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 39.6 Å / Resolution method: FSC 0.5 CUT-OFF / Number subtomograms used: 2200
ExtractionNumber tomograms: 10 / Number images used: 2400
Final angle assignmentType: NOT APPLICABLE

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