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- EMDB-6312: Ciliary microtubule doublet by single particle analysis -

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Basic information

Entry
Database: EMDB / ID: EMD-6312
TitleCiliary microtubule doublet by single particle analysis
Map dataCiliary microtubule doublet
Sample
  • Sample: microtubule doublet from Tetrahymena cilia
  • Organelle or cellular component: cilium
Keywordscilia / microtubule / tubulin / dynein
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / microtubule / hydrolase activity / GTPase activity / GTP binding
Similarity search - Function
Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain ...Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Tubulin alpha chain / Tubulin beta chain
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 23.0 Å
AuthorsMaheshwari A / Obbineni JM / Bui KH / Shibata K / Toyoshima Y / Ishikawa T
CitationJournal: Structure / Year: 2015
Title: α- and β-Tubulin Lattice of the Axonemal Microtubule Doublet and Binding Proteins Revealed by Single Particle Cryo-Electron Microscopy and Tomography.
Authors: Aditi Maheshwari / Jagan Mohan Obbineni / Khanh Huy Bui / Keitaro Shibata / Yoko Y Toyoshima / Takashi Ishikawa /
Abstract: Microtubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the ...Microtubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the reconstituted microtubule has been solved at atomic resolution, nature of the axonemal MTD is still unclear. There are a few hypotheses of the lattice arrangement of its α- and β-tubulins, but it has not been described how dyneins and radial spokes bind to MTD. In this study, we analyzed the three-dimensional structure of Tetrahymena MTD at ∼19 Å resolution by single particle cryo-electron microscopy. To identify α- and β-tubulins, we combined image analysis of MTD with specific kinesin decoration. This work reveals that α- and β-tubulins form a B-lattice arrangement in the entire MTD with a seam at the outer junction. We revealed the unique way in which inner arm dyneins, radial spokes, and proteins inside MTD bind and bridge protofilaments.
History
DepositionMar 27, 2015-
Header (metadata) releaseApr 8, 2015-
Map releaseAug 5, 2015-
UpdateSep 9, 2015-
Current statusSep 9, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jao
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jao
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6312.gif

Downloads & links

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Map

FileDownload / File: emd_6312.map.gz / Format: CCP4 / Size: 124.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCiliary microtubule doublet
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.48 Å/pix.
x 322 pix.
= 1441.916 Å		4.48 Å/pix.
x 322 pix.
= 1441.916 Å		4.48 Å/pix.
x 322 pix.
= 1441.916 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.478 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 1.3 / Movie #1: 1.3
Minimum - Maximum-33.726356510000002 - 33.660144809999998
Average (Standard dev.)0.09271099 (±2.29999352)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions322322322
Spacing322322322
CellA=B=C: 1441.916 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.4784.4784.478
M x/y/z322322322
origin x/y/z0.0000.0000.000
length x/y/z1441.9161441.9161441.916
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS322322322
D min/max/mean-33.72633.6600.093

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Supplemental data

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Sample components

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Entire : microtubule doublet from Tetrahymena cilia

EntireName: microtubule doublet from Tetrahymena cilia
Components
  • Sample: microtubule doublet from Tetrahymena cilia
  • Organelle or cellular component: cilium

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Supramolecule #1000: microtubule doublet from Tetrahymena cilia

SupramoleculeName: microtubule doublet from Tetrahymena cilia / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: cilium

SupramoleculeName: cilium / type: organelle_or_cellular_component / ID: 1 / Name.synonym: flagellum
Details: Microtubule doublets were prepared from Tetrahymena cilia.
Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB210 / Organelle: cilium

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
Details: 30 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, 4 mM CaCl2
GridDetails: 300 mesh copper grid with holey carbon film, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK II / Method: Blot for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 80 K / Max: 100 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
Specialist opticsEnergy filter - Name: GIF Trideem / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
DateOct 25, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.5 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 67000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.0 Å
Applied symmetry - Helical parameters - Δ&Phi: 28.0 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: Spider / Number images used: 10700

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Atomic model buiding 1

Initial modelPDB ID:

3j6e

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation
Output model

PDB-3jao:
Ciliary microtubule doublet

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