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- EMDB-5850: Cryo-electron tomography of Chlamydomonas axoneme labeled with st... -

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Basic information

Entry
Database: EMDB / ID: EMD-5850
TitleCryo-electron tomography of Chlamydomonas axoneme labeled with streptavidin (RSP6 N-terminus tag)
Map dataAveraged tomogram of axonemal doublet microtubules (RSP6 N-terminus tag)
Sample
  • Sample: Chlamydomonas axoneme, RSP6 BCCP N-terminus tag
  • Organelle or cellular component: Axoneme
KeywordsCryo-electron tomography / Chlamydomonas reinhardtii / biotin-streptavidin / cilia and flagella / dynein / structural labeling / radial spoke
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 71.0 Å
AuthorsOda T / Yanagisawa H / Yagi T / Kikkawa M
CitationJournal: J Cell Biol / Year: 2014
Title: Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity.
Authors: Toshiyuki Oda / Haruaki Yanagisawa / Toshiki Yagi / Masahide Kikkawa /
Abstract: Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the ...Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.
History
DepositionJan 6, 2014-
Header (metadata) releaseFeb 19, 2014-
Map releaseFeb 19, 2014-
UpdateMay 7, 2014-
Current statusMay 7, 2014Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 34398
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 34398
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5850_1.tif

Downloads & links

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Map

FileDownload / File: emd_5850.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAveraged tomogram of axonemal doublet microtubules (RSP6 N-terminus tag)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.07 Å/pix.
x 240 pix.
= 1456.8 Å		6.07 Å/pix.
x 240 pix.
= 1456.8 Å		6.07 Å/pix.
x 240 pix.
= 1456.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 34398.0 / Movie #1: 34398
Minimum - Maximum0.00205192 - 39462.3671875
Average (Standard dev.)10783.844726560000709 (±14642.99609375)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 1456.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.076.076.07
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z1456.8001456.8001456.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean0.00239462.36710783.845

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Supplemental data

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Sample components

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Entire : Chlamydomonas axoneme, RSP6 BCCP N-terminus tag

EntireName: Chlamydomonas axoneme, RSP6 BCCP N-terminus tag
Components
  • Sample: Chlamydomonas axoneme, RSP6 BCCP N-terminus tag
  • Organelle or cellular component: Axoneme

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Supramolecule #1000: Chlamydomonas axoneme, RSP6 BCCP N-terminus tag

SupramoleculeName: Chlamydomonas axoneme, RSP6 BCCP N-terminus tag / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Axoneme

SupramoleculeName: Axoneme / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: 137c / synonym: green algae / 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

Concentration0.01 mg/mL
BufferpH: 7.2
Details: 30 mM Hepes-NaOH pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, and 50 mM NaCl
GridDetails: 300 mesh copper grid with home-made holey carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: LEICA EM GP / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3100FFC
TemperatureMin: 90 K / Max: 95 K
Specialist opticsEnergy filter - Name: JEOL Omega / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 23.0 eV
DateSep 3, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 90 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 25700 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal magnification: 15000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60.5 ° / Tilt series - Axis1 - Max angle: 60.5 °

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 71.0 Å / Resolution method: OTHER / Software - Name: PEET, RUBY-HELIX / Number subtomograms used: 502

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