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- EMDB-9618: Localization of FAP70 N-terminus on Chlamydomonas axoneme -

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Basic information

Entry
Database: EMDB / ID: EMD-9618
TitleLocalization of FAP70 N-terminus on Chlamydomonas axoneme
Map datafap70::FAP70-N-BCCP Streptavidin labeled
Sample
  • Organelle or cellular component: Streptavidin labeled fap70::FAP70-N-BCCP axoneme
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 40.0 Å
AuthorsShamoto N / Narita K / Kubo T / Oda T / Takeda S
CitationJournal: Cells / Year: 2018
Title: CFAP70 Is a Novel Axoneme-Binding Protein That Localizes at the Base of the Outer Dynein Arm and Regulates Ciliary Motility.
Authors: Noritoshi Shamoto / Keishi Narita / Tomohiro Kubo / Toshiyuki Oda / Sen Takeda /
Abstract: In the present study, we characterized CFAP70, a candidate of cilia-related protein in mice. As this protein has a cluster of tetratricopeptide repeat (TPR) domains like many components of the ...In the present study, we characterized CFAP70, a candidate of cilia-related protein in mice. As this protein has a cluster of tetratricopeptide repeat (TPR) domains like many components of the intraflagellar transport (IFT) complex, we investigated the domain functions of particular interest in ciliary targeting and/or localization. RT-PCR and immunohistochemistry of various mouse tissues demonstrated the association of CFAP70 with motile cilia and flagella. A stepwise extraction of proteins from swine tracheal cilia showed that CFAP70 bound tightly to the ciliary axoneme. Fluorescence microscopy of the cultured ependyma expressing fragments of CFAP70 demonstrated that the N-terminus rather than the C-terminus with the TPR domains was more important for the ciliary localization. When CFAP70 was knocked down in cultured mouse ependyma, reductions in cilia beating frequency were observed. Consistent with these observations, a mutant lacking the CFAP70 homolog, FAP70, showed defects in outer dynein arm (ODA) activity and a reduction in flagellar motility. Cryo-electron tomography revealed that the N-terminus of FAP70 resided stably at the base of the ODA. These results demonstrated that CFAP70 is a novel regulatory component of the ODA in motile cilia and flagella, and that the N-terminus is important for its ciliary localization.
History
DepositionAug 14, 2018-
Header (metadata) releaseSep 12, 2018-
Map releaseSep 12, 2018-
UpdateSep 12, 2018-
Current statusSep 12, 2018Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 557
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 557
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9618.png

Downloads & links

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Map

FileDownload / File: emd_9618.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfap70::FAP70-N-BCCP Streptavidin labeled
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.03 Å/pix.
x 200 pix.
= 1405. Å		7.03 Å/pix.
x 200 pix.
= 1405. Å		7.03 Å/pix.
x 200 pix.
= 1405. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7.025 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 557. / Movie #1: 557
Minimum - Maximum0. - 1055.426400000000058
Average (Standard dev.)137.692370000000011 (±220.180470000000014)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 1405.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.0257.0257.025
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z1405.0001405.0001405.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-69-50-137
NX/NY/NZ136114193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean0.0001055.426137.692

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Supplemental data

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Sample components

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Entire : Streptavidin labeled fap70::FAP70-N-BCCP axoneme

EntireName: Streptavidin labeled fap70::FAP70-N-BCCP axoneme
Components
  • Organelle or cellular component: Streptavidin labeled fap70::FAP70-N-BCCP axoneme

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Supramolecule #1: Streptavidin labeled fap70::FAP70-N-BCCP axoneme

SupramoleculeName: Streptavidin labeled fap70::FAP70-N-BCCP axoneme / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Chlamydomonas axoneme expressing FAP70-N-BCCP labeled with streptavidin and biotinylated cytochrome c
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: cc125 / Organelle: Flagella

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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: 7.2
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot 5.5 sec before plunging.

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Electron microscopy

MicroscopeJEOL 3100FEF
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 40 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 6.0 sec. / Average electron dose: 1.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: GATAN 914 HIGH TILT LIQUID NITROGEN CRYO TRANSFER TOMOGRAPHY HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET / Number subtomograms used: 1000
ExtractionNumber tomograms: 9 / Number images used: 1448 / Software - Name: PEET
CTF correctionSoftware - Name: IMOD
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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