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- EMDB-5330: Cryo-electron tomography reveals novel interactions and doublet-s... -

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Basic information

Entry
Database: EMDB / ID: EMD-5330
TitleCryo-electron tomography reveals novel interactions and doublet-specific structures in the I1 dynein
Map dataThis is a subtomogram average of the I1 inner dynein complex in wild type Chlamydomonas flagella
Sample
  • Sample: Cryo-electron tomography and subtomographic average (750 axonemal repeats) of isolated axonemes of wild type Chlamydomonas (CC 125, 137c), I1 dynein complex (dynein f) is bound to the doublet microtubule and is connected to neighboring structures.
  • Organelle or cellular component: I1 dynein complex
Keywordsaxoneme / molecular motor / motility regulation / flagella
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 39.0 Å
AuthorsHeuser T / Barber CF / Lin J / Krell J / Rebesco M / Porter ME / Nicastro D
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein.
Authors: Thomas Heuser / Cynthia F Barber / Jianfeng Lin / Jeremy Krell / Matthew Rebesco / Mary E Porter / Daniela Nicastro /
Abstract: Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a ...Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility.
History
DepositionAug 10, 2011-
Header (metadata) releaseAug 11, 2011-
Map releaseJun 21, 2012-
UpdateApr 2, 2014-
Current statusApr 2, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 127.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 127.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5330_1.tif

Downloads & links

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Map

FileDownload / File: emd_5330.map.gz / Format: CCP4 / Size: 336.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a subtomogram average of the I1 inner dynein complex in wild type Chlamydomonas flagella
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
9.5 Å/pix.
x 40 pix.
= 380. Å		9.5 Å/pix.
x 40 pix.
= 522.5 Å		9.5 Å/pix.
x 55 pix.
= 380. 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: 9.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 127.5 / Movie #1: 127.5
Minimum - Maximum109.595199579999999 - 148.880630489999987
Average (Standard dev.)124.201629639999993 (±5.68234873)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-53-21-64
Dimensions405540
Spacing554040
CellA: 380.0 Å / B: 522.5 Å / C: 380.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z9.59.59.5
M x/y/z405540
origin x/y/z0.0000.0000.000
length x/y/z380.000522.500380.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-21-53-64
NC/NR/NS554040
D min/max/mean109.595148.881124.202

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Supplemental data

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Sample components

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Entire : Cryo-electron tomography and subtomographic average (750 axonemal...

EntireName: Cryo-electron tomography and subtomographic average (750 axonemal repeats) of isolated axonemes of wild type Chlamydomonas (CC 125, 137c), I1 dynein complex (dynein f) is bound to the doublet ...Name: Cryo-electron tomography and subtomographic average (750 axonemal repeats) of isolated axonemes of wild type Chlamydomonas (CC 125, 137c), I1 dynein complex (dynein f) is bound to the doublet microtubule and is connected to neighboring structures.
Components
  • Sample: Cryo-electron tomography and subtomographic average (750 axonemal repeats) of isolated axonemes of wild type Chlamydomonas (CC 125, 137c), I1 dynein complex (dynein f) is bound to the doublet microtubule and is connected to neighboring structures.
  • Organelle or cellular component: I1 dynein complex

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Supramolecule #1000: Cryo-electron tomography and subtomographic average (750 axonemal...

SupramoleculeName: Cryo-electron tomography and subtomographic average (750 axonemal repeats) of isolated axonemes of wild type Chlamydomonas (CC 125, 137c), I1 dynein complex (dynein f) is bound to the doublet ...Name: Cryo-electron tomography and subtomographic average (750 axonemal repeats) of isolated axonemes of wild type Chlamydomonas (CC 125, 137c), I1 dynein complex (dynein f) is bound to the doublet microtubule and is connected to neighboring structures.
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: I1 dynein complex

SupramoleculeName: I1 dynein complex / type: organelle_or_cellular_component / ID: 1 / Name.synonym: dynein f / Number of copies: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: CC-125, 137c / synonym: unicellular green algae / Cell: Chlamydomonas reinhardtii / Organelle: eukaryotic flagella

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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: 10 mM HEPES, pH 7.4, 25 mM NaCl, 4 mM MgSO4, 1 mM EGTA, 0.1 mM EDTA
GridDetails: Quantifoil holey carbon grids Cu 200 mesh R2/2
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Method: front-side blotting for 2-3 seconds

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 80 K
Specialist opticsEnergy filter - Name: GATAN postcolumn filter GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateFeb 20, 2004
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (2k x 2k) / Average electron dose: 100 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 13500
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Details750 axonemal repeats (96 nm long) from 5 tomograms (reconstructed using fiducial alignment and weighted backprojection, IMOD software, Kremer et al. 1996) were aligned and averaged using the PEET software (bio3d.colorado.edu, Nicastro et al. 2006). Average number of tilts used in the 3D reconstructions: 80. Average tomographic tilt angle increment: 1.5.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 39.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD
Details: Final maps were calculated by averaging 750 particles from 5 tomograms

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