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- EMDB-2129: Cryo-electron tomography averaged map of microtubule doublet 9 in... -

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Basic information

Entry
Database: EMDB / ID: EMD-2129
TitleCryo-electron tomography averaged map of microtubule doublet 9 in the proximal region of Chlamydomonas axoneme
Map dataReconstruction of outer doublet 9 of the Chlamydomonas axoneme in the proximal region.
Sample
  • Sample: Outer doublet 9 of Chlamydomonas axoneme in the proximal region
  • Organelle or cellular component: flagellum
Keywordsaxoneme / dynein / Chlamydomonas / microtubule doublet
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 42.9 Å
AuthorsBui KH / Yagi T / Yamamoto R / Kamiya R / Ishikawa T
CitationJournal: J Cell Biol / Year: 2012
Title: Polarity and asymmetry in the arrangement of dynein and related structures in the Chlamydomonas axoneme.
Authors: Khanh Huy Bui / Toshiki Yagi / Ryosuke Yamamoto / Ritsu Kamiya / Takashi Ishikawa /
Abstract: Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet ...Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.
History
DepositionJun 11, 2012-
Header (metadata) releaseJun 13, 2012-
Map releaseSep 12, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2129.png

Downloads & links

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Map

FileDownload / File: emd_2129.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of outer doublet 9 of the Chlamydomonas axoneme in the proximal region.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.25 Å/pix.
x 200 pix.
= 1450. Å		7.25 Å/pix.
x 200 pix.
= 1450. Å		7.25 Å/pix.
x 200 pix.
= 1450. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7.25 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-6.53235579 - 9.76999664
Average (Standard dev.)0.00000002 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 1450.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.257.257.25
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z1450.0001450.0001450.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-6.5329.7700.000

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Supplemental data

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Sample components

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Entire : Outer doublet 9 of Chlamydomonas axoneme in the proximal region

EntireName: Outer doublet 9 of Chlamydomonas axoneme in the proximal region
Components
  • Sample: Outer doublet 9 of Chlamydomonas axoneme in the proximal region
  • Organelle or cellular component: flagellum

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Supramolecule #1000: Outer doublet 9 of Chlamydomonas axoneme in the proximal region

SupramoleculeName: Outer doublet 9 of Chlamydomonas axoneme in the proximal region
type: sample / ID: 1000 / Details: Flagella were isolated from Chlamydomonas. / Number unique components: 1

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Supramolecule #1: flagellum

SupramoleculeName: flagellum / type: organelle_or_cellular_component / ID: 1 / Name.synonym: axoneme,cilia / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: c137 (mt+) / synonym: green algae / Organelle: flagellum / Location in cell: proximal part of the flagella

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.4
Details: 30 mM Hepes, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.5% (wt/vol) polyethylene glycol (MW 20,000)
GridDetails: 300 mesh Quantifoil Holey Carbon copper grid R2/1
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 110 K / Instrument: FEI VITROBOT MARK II / Method: Offset -3, blot 3 seconds, drain time 0 second

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 93 K / Max: 118 K / Average: 98 K
Specialist opticsEnergy filter - Name: Gatan Tridem / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateNov 26, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 499 / Average electron dose: 60 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 19303 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 27500
Sample stageSpecimen holder: Gatan 626, liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °

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Image processing

DetailsR-weighted back projection using IMOD with fiducial markers. Average number of tilts used in the 3D reconstructions: 61. Average tomographic tilt angle increment: 2.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 42.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD, BSOFT, SPIDER, TOM, package, Matlab

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