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- EMDB-6874: A microtubule-dynein tethering complex regulates the axonemal inn... -

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Basic information

Entry
Database: EMDB / ID: EMD-6874
TitleA microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)
Map dataChlamydomonas axoneme, FAP44-C-BCCP labeled with streptavidin
Sample
  • Organelle or cellular component: Axoneme, outer doublet microtubule
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 60.0 Å
AuthorsKubo T / Hou Y / Cochran DA / Witman GB / Oda T
CitationJournal: Mol Biol Cell / Year: 2018
Title: A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1).
Authors: Tomohiro Kubo / Yuqing Hou / Deborah A Cochran / George B Witman / Toshiyuki Oda /
Abstract: Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but ...Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.
History
DepositionDec 24, 2017-
Header (metadata) releaseMar 28, 2018-
Map releaseMar 28, 2018-
UpdateMar 28, 2018-
Current statusMar 28, 2018Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 189
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 189
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6874.png

Downloads & links

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Map

FileDownload / File: emd_6874.map.gz / Format: CCP4 / Size: 9.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationChlamydomonas axoneme, FAP44-C-BCCP labeled with streptavidin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8 Å/pix.
x 111 pix.
= 888. Å		8 Å/pix.
x 192 pix.
= 1536. Å		8 Å/pix.
x 116 pix.
= 928. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 189. / Movie #1: 189
Minimum - Maximum0. - 386.908799999999985
Average (Standard dev.)95.038460000000001 (±92.448524000000006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0-28-30
Dimensions192116111
Spacing116192111
CellA: 928.0 Å / B: 1536.0 Å / C: 888.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z888
M x/y/z116192111
origin x/y/z0.0000.0000.000
length x/y/z928.0001536.000888.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-280-30
NC/NR/NS116192111
D min/max/mean0.000386.90995.038

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Supplemental data

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Sample components

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Entire : Axoneme, outer doublet microtubule

EntireName: Axoneme, outer doublet microtubule
Components
  • Organelle or cellular component: Axoneme, outer doublet microtubule

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Supramolecule #1: Axoneme, outer doublet microtubule

SupramoleculeName: Axoneme, outer doublet microtubule / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Details: FAP44-C-BCCP axoneme labeled with streptavidin
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: fap44:FAP44-C-BCCP / 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: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
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 2100F
Image recordingFilm or detector model: TVIPS TEMCAM-F216 (2k x 2k) / Detector mode: COUNTING / Average exposure time: 0.2 sec. / Average electron dose: 1.6 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER 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: 60.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number subtomograms used: 1197
ExtractionNumber tomograms: 9 / Number images used: 1300
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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