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- EMDB-5252: electron cryo-tomography reconstruction and subvolume averaging o... -

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Basic information

Entry
Database: EMDB / ID: EMD-5252
Titleelectron cryo-tomography reconstruction and subvolume averaging of the basal body triplet from Chlamydomonas reinhardtii
Map dataaveraged microtubule triplet from basal body from Chlamydomonas reinhardtii
Sample
  • Sample: basal body microtubule triplet
  • Organelle or cellular component: basal body
Keywordsbasal body / centriole / electron cryo-tomography / three-dimensional reconstruction
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 33.0 Å
AuthorsLi S / Fernandez JJ / Marshall WF / Agard DA
CitationJournal: EMBO J / Year: 2012
Title: Three-dimensional structure of basal body triplet revealed by electron cryo-tomography.
Authors: Sam Li / Jose-Jesus Fernandez / Wallace F Marshall / David A Agard /
Abstract: Basal bodies and centrioles play central roles in microtubule (MT)-organizing centres within many eukaryotes. They share a barrel-shaped cylindrical structure composed of nine MT triplet blades. ...Basal bodies and centrioles play central roles in microtubule (MT)-organizing centres within many eukaryotes. They share a barrel-shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo-tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo-atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non-tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ-tubulin and other components participate in the assembly of the basal body.
History
DepositionDec 8, 2010-
Header (metadata) releaseJun 23, 2011-
Map releaseFeb 27, 2012-
UpdateOct 3, 2012-
Current statusOct 3, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 146
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 146
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5252_1.tif

Downloads & links

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Map

FileDownload / File: emd_5252.map.gz / Format: CCP4 / Size: 6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationaveraged microtubule triplet from basal body from Chlamydomonas reinhardtii
Voxel sizeX=Y=Z: 6.5 Å
Density
Contour LevelBy AUTHOR: 146.0 / Movie #1: 146
Minimum - Maximum33.072956089999998 - 288.799804689999974
Average (Standard dev.)127.000007629999999 (±14.999806400000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions20020040
Spacing20020040
CellA: 1300.0 Å / B: 1300.0 Å / C: 260.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.56.56.5
M x/y/z20020040
origin x/y/z0.0000.0000.000
length x/y/z1300.0001300.000260.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS20020040
D min/max/mean33.073288.800127.000

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Supplemental data

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Sample components

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Entire : basal body microtubule triplet

EntireName: basal body microtubule triplet
Components
  • Sample: basal body microtubule triplet
  • Organelle or cellular component: basal body

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Supramolecule #1000: basal body microtubule triplet

SupramoleculeName: basal body microtubule triplet / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 15.1 MDa
Method: estimation based on the volume assuming the protein density is 1.41g per cm3

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Supramolecule #1: basal body

SupramoleculeName: basal body / type: organelle_or_cellular_component / ID: 1 / Name.synonym: basal body / Number of copies: 2 / Oligomeric state: singlet / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: cc3491 / synonym: green algea / Cell: Chlamydomonas reinhardtii / Organelle: basal body / Location in cell: flagellum apparatus

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging

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Sample preparation

BufferpH: 8 / Details: 10mM Tris 1mM EDTA
GridDetails: 300 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 46153 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus min: 9.0 µm / Nominal magnification: 34000
Specialist opticsEnergy filter - Name: Gatan GIF2000 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
TemperatureMin: 91 K / Max: 93 K / Average: 93 K
Detailsdefocus maximum value is 24000nm
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 80 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image, Wiener filter
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 33.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Priism / Details: Final map was average of 1644 subvolumes
DetailsThe tomographic tilt series were taken by UCSF Tomography program. Average number of tilts used in the 3D reconstructions: 82. Average tomographic tilt angle increment: 2.

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Atomic model buiding 1

Initial modelPDB ID:

1jff

SoftwareName: Chimera
DetailsProtocol: rigid body. The atomic structure was first manually fitting into EM density by program O and the fitting was refined by Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation coefficiet

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