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- EMDB-3557: Centriolar Distal MT doublets in centrosome extracted from o.arie... -

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Basic information

Entry
Database: EMDB / ID: EMD-3557
TitleCentriolar Distal MT doublets in centrosome extracted from o.aries thymocytes
Map dataMT doublet at the distal part of the centriolar wall of centrosome in mammals. The subtomogram averaging was processed using tomograms of centrosomes extracted from o.aries thymocytes
Sample
  • Organelle or cellular component: Centrosomes enriched from young lamb thymocyte
    • Organelle or cellular component: Centriole
Biological speciesOvis aries (sheep)
Methodsubtomogram averaging / cryo EM / Resolution: 46.7 Å
AuthorsBusselez J / Chichon FJ / Melero R / Carrascosa JL / Carazo JM
CitationJournal: Sci Rep / Year: 2019
Title: Cryo-Electron Tomography and Proteomics studies of centrosomes from differentiated quiescent thymocytes.
Authors: Johan Busselez / Francisco Javier Chichón / Maria Josefa Rodríguez / Adan Alpízar / Séverine Isabelle Gharbi / Mònica Franch / Roberto Melero / Alberto Paradela / José L Carrascosa / José-Maria Carazo /
Abstract: We have used cryo Electron Tomography, proteomics and immunolabeling to study centrosomes isolated from the young lamb thymus, an efficient source of quiescent differentiated cells. We compared the ...We have used cryo Electron Tomography, proteomics and immunolabeling to study centrosomes isolated from the young lamb thymus, an efficient source of quiescent differentiated cells. We compared the proteome of thymocyte centrosomes to data published for KE37 cells, focusing on proteins associated with centriole disengagement and centrosome separation. The data obtained enhances our understanding of the protein system joining the centrioles, a system comprised of a branched network of fibers linked to an apparently amorphous density that was partially characterized here. A number of proteins were localized to the amorphous density by immunolabeling (C-NAP1, cohesin SMC1, condensin SMC4 and NCAPD2), yet not DNA. In conjuction, these data not only extend our understanding of centrosomes but they will help refine the model that focus on the protein system associated with the centriolar junction.
History
DepositionDec 24, 2016-
Header (metadata) releaseJan 25, 2017-
Map releaseAug 22, 2018-
UpdateAug 22, 2018-
Current statusAug 22, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3557.png

Downloads & links

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Map

FileDownload / File: emd_3557.map.gz / Format: CCP4 / Size: 61 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMT doublet at the distal part of the centriolar wall of centrosome in mammals. The subtomogram averaging was processed using tomograms of centrosomes extracted from o.aries thymocytes
Voxel sizeX=Y=Z: 3.42 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum-4.712067 - 7.2480493
Average (Standard dev.)0.05829562 (±0.5349598)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions252252252
Spacing252252252
CellA=B=C: 861.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z252252252
origin x/y/z0.0000.0000.000
length x/y/z861.840861.840861.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS252252252
D min/max/mean-4.7127.2480.058

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Supplemental data

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Sample components

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Entire : Centrosomes enriched from young lamb thymocyte

EntireName: Centrosomes enriched from young lamb thymocyte
Components
  • Organelle or cellular component: Centrosomes enriched from young lamb thymocyte
    • Organelle or cellular component: Centriole

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Supramolecule #1: Centrosomes enriched from young lamb thymocyte

SupramoleculeName: Centrosomes enriched from young lamb thymocyte / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: The centrosomes were extracted accordingly to Komesli et. al. 1989
Source (natural)Organism: Ovis aries (sheep) / Organ: Thymus / Tissue: Thymus Lobules / Organelle: Centrosome / Location in cell: cytoplasm near the nucleus
Molecular weightExperimental: 230000 MDa

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Supramolecule #2: Centriole

SupramoleculeName: Centriole / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Ovis aries (sheep) / Organ: Thymus / Tissue: Thymus Lobules / Organelle: Centrosome / Location in cell: cytoplasm near the nucleus

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.076 mg/mL
BufferpH: 7.2 / Component - Concentration: 10.0 mM / Component - Formula: C8H18N2O6S2 / Component - Name: Pipes / Details: pH Rectified with KOH
GridModel: quantifoil 3.5/1 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: FORMVAR / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC
DetailsThe sample was enriched by two step of sedimentation in sucrose gradient. The sucrose was washed out in several drops of buffer, the last of which included fiducial markers (10 nm bovine serum albumin (BSA)-coated gold beads) for tilt series alignment

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 5.906 µm / Calibrated defocus min: 5.54 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: Gatan 968 Quantum / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average exposure time: 1.0 sec. / Average electron dose: 1.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 861.64 Å / Unit cell - B: 861.64 Å / Unit cell - C: 249.66 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: P1
ExtractionNumber tomograms: 4 / Number images used: 1111 / Method: volume picked interactively / Software - Name: Dynamo (ver. 1.1.22)
Details: coarse models of the centriole microtubules were obtained using Fiber Tracing in Amira and converted for import in dynamo. from those coarse models, "filament with torsion" models were ...Details: coarse models of the centriole microtubules were obtained using Fiber Tracing in Amira and converted for import in dynamo. from those coarse models, "filament with torsion" models were traced in dynamo. the doublets were segregated from the triplets and doublets volume particles were windowed with overlap every 8.3nm along the filament with torsion models
CTF correctionSoftware - Name: TOMOCTF / Details: wiener filtering, made on tilt series
Final 3D classificationNumber classes: 4 / Software - Name: Dynamo / Details: classification by PCA+k-means
Final angle assignmentType: OTHER / Software - Name: Dynamo (ver. 1.1.22)
Details: The particular geometry of the centriole with its repeat every 8.3nm along the proximal-distal axis and its coarse 9 fold symmetry was used to construct from each tomogram a coarse model ...Details: The particular geometry of the centriole with its repeat every 8.3nm along the proximal-distal axis and its coarse 9 fold symmetry was used to construct from each tomogram a coarse model lowly affected by missing wedge. Those coarse models where volume aligned and a initial reference were averaged from all the subtomogram particle using the coarse orientation of the particles determined in that way. This model was iteratively refined keeping for each iteration the particle with the 70% best cross-correlation to average the reference for the next-iteration. For the finer step a mask was used to restrict the alignment to 3 8.3nm repeats.
Final reconstructionNumber classes used: 2 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 46.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Dynamo (ver. 1.1.22) / Number subtomograms used: 413
DetailsTilt series were aligned based on fiducials with IMOD and mass normalized using PRIISM. The ctf correction was processed on tilt series using TomoCTF. Tomogram were reconstructed using SIRT algorithm with TOMO3D. The number of frames per image were variable, with 8 frames at full tilt, through to 4 frames at zero tilt.
FSC plot (resolution estimation)

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