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- EMDB-1941: Radial Spoke from Chlamydomonas flagella -

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Basic information

Entry
Database: EMDB / ID: EMD-1941
TitleRadial Spoke from Chlamydomonas flagella
Map dataThis is a density map of Radial Spokes from Chlamydomonas flagella.
Sample
  • Sample: Radial spoke from flagella/cilia
  • Organelle or cellular component: radial spoke
Keywordsradial spoke / flagella / cilia / Chlamydomonas
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 39.18 Å
AuthorsPigino G / Bui KH / Maheshwari A / Lupetti P / Diener D / Ishikawa T
CitationJournal: J Cell Biol / Year: 2011
Title: Cryoelectron tomography of radial spokes in cilia and flagella.
Authors: Gaia Pigino / Khanh Huy Bui / Aditi Maheshwari / Pietro Lupetti / Dennis Diener / Takashi Ishikawa /
Abstract: Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 ...Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 polypeptides, whose interactions and placement must be deciphered to understand RS function. In this paper, we show the detailed three-dimensional (3D) structure of RS in situ in Chlamydomonas reinhardtii flagella and Tetrahymena thermophila cilia that we obtained using cryoelectron tomography (cryo-ET). We clarify similarities and differences between the three spoke species, RS1, RS2, and RS3, in T. thermophila and in C. reinhardtii and show that part of RS3 is conserved in C. reinhardtii, which only has two species of complete RSs. By analyzing C. reinhardtii mutants, we identified the specific location of subsets of RS proteins (RSPs). Our 3D reconstructions show a twofold symmetry, suggesting that fully assembled RSs are produced by dimerization. Based on our cryo-ET data, we propose models of subdomain organization within the RS as well as interactions between RSPs and with other axonemal components.
History
DepositionJul 30, 2011-
Header (metadata) releaseOct 20, 2011-
Map releaseOct 20, 2011-
UpdateAug 29, 2012-
Current statusAug 29, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1941.png

Downloads & links

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Map

FileDownload / File: emd_1941.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a density map of Radial Spokes from Chlamydomonas flagella.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.78 Å/pix.
x 143 pix.
= 969.54 Å		6.78 Å/pix.
x 43 pix.
= 291.54 Å		6.78 Å/pix.
x 89 pix.
= 603.42 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: 6.78 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-7.84166 - 17.269300000000001
Average (Standard dev.)0.58137 (±2.46525)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin191413
Dimensions4389143
Spacing4389143
CellA: 603.42 Å / B: 291.54 Å / C: 969.54 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.786.786.78
M x/y/z8943143
origin x/y/z0.0000.0000.000
length x/y/z603.420291.540969.540
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS141913
NC/NR/NS8943143
D min/max/mean-7.84217.2690.581

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Supplemental data

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Sample components

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Entire : Radial spoke from flagella/cilia

EntireName: Radial spoke from flagella/cilia
Components
  • Sample: Radial spoke from flagella/cilia
  • Organelle or cellular component: radial spoke

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Supramolecule #1000: Radial spoke from flagella/cilia

SupramoleculeName: Radial spoke from flagella/cilia / type: sample / ID: 1000 / Details: Radial spokes from Axoneme / Number unique components: 1

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Supramolecule #1: radial spoke

SupramoleculeName: radial spoke / type: organelle_or_cellular_component / ID: 1 / Name.synonym: radial spoke / Recombinant expression: Yes
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: 137c / synonym: Chlamydomonas / Tissue: flagella / Cell: 137c / Organelle: flagella / Location in cell: flagella
Recombinant expressionOrganism: Chlamydomonas reinhardtii (plant)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Details: HMDKP
GridDetails: lacey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: 3 second blotting

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 100 K / Max: 100 K / Average: 100 K
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 0.6 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 19000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.5 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 27500
Sample stageSpecimen holder: Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °

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Image processing

DetailsSubtomograms were averaged. Average number of tilts used in the 3D reconstructions: 61. Average tomographic tilt angle increment: 2.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 39.18 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD

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