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- EMDB-19781: Cryo-ET of a mitotic centrosome in an embryonic C. elegans cell -

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Basic information

Entry
Database: EMDB / ID: EMD-19781
TitleCryo-ET of a mitotic centrosome in an embryonic C. elegans cell
Map dataRepresentative C. elegans centrosome tomogram
Sample
  • Cell: Dissociated C. elegans embryonic cells
Keywordscryo-ET / cryo-FIB / C. elegans / mitosis / centrosome / centriole / microtubules / CELL CYCLE
Biological speciesCaenorhabditis elegans (invertebrata)
Methodelectron tomography / cryo EM
AuthorsTollervey F / Rios MU / Zagoriy I / Woodruff JB / Mahamid J
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)760067European Union
CitationJournal: bioRxiv / Year: 2024
Title: Native molecular architectures of centrosomes in embryos.
Authors: Fergus Tollervey / Manolo U Rios / Evgenia Zagoriy / Jeffrey B Woodruff / Julia Mahamid /
Abstract: Centrosomes organize microtubules that are essential for mitotic divisions in animal cells. They consist of centrioles surrounded by Pericentriolar Material (PCM). Questions related to mechanisms of ...Centrosomes organize microtubules that are essential for mitotic divisions in animal cells. They consist of centrioles surrounded by Pericentriolar Material (PCM). Questions related to mechanisms of centriole assembly, PCM organization, and microtubule formation remain unanswered, in part due to limited availability of molecular-resolution structural analyses . Here, we use cryo-electron tomography to visualize centrosomes across the cell cycle in cells isolated from embryos. We describe a pseudo-timeline of centriole assembly and identify distinct structural features including a cartwheel in daughter centrioles, and incomplete microtubule doublets surrounded by a star-shaped density in mother centrioles. We find that centriole and PCM microtubules differ in protofilament number (13 versus 11) indicating distinct nucleation mechanisms. This difference could be explained by atypical γ-tubulin ring complexes with 11-fold symmetry identified at the minus ends of short PCM microtubules. We further characterize a porous and disordered network that forms the interconnected PCM. Thus, our work builds a three-dimensional structural atlas that helps explain how centrosomes assemble, grow, and achieve function.
History
DepositionMar 3, 2024-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19781.png

Downloads & links

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Map

FileDownload / File: emd_19781.map.gz / Format: CCP4 / Size: 1.7 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRepresentative C. elegans centrosome tomogram
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.48 Å/pix.
x 500 pix.
= 6740.4 Å		13.48 Å/pix.
x 928 pix.
= 12510.182 Å		13.48 Å/pix.
x 960 pix.
= 12941.567 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 13.4808 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.84275424 - 0.7648583
Average (Standard dev.)-0.000005652757 (±0.04591443)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions928960500
Spacing960928500
CellA: 12941.567 Å / B: 12510.182 Å / C: 6740.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Binary segmentation of centrioles

Fileemd_19781_additional_1.map
AnnotationBinary segmentation of centrioles
Projections & Slices
AxesZYX

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Additional map: Binary segmentation of microtubules

Fileemd_19781_additional_2.map
AnnotationBinary segmentation of microtubules
Projections & Slices
AxesZYX

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Additional map: Binary segmentation of ribosomes

Fileemd_19781_additional_3.map
AnnotationBinary segmentation of ribosomes
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Binary segmentation of ribosomes

Fileemd_19781_additional_4.map
AnnotationBinary segmentation of ribosomes
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dissociated C. elegans embryonic cells

EntireName: Dissociated C. elegans embryonic cells
Components
  • Cell: Dissociated C. elegans embryonic cells

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Supramolecule #1: Dissociated C. elegans embryonic cells

SupramoleculeName: Dissociated C. elegans embryonic cells / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Caenorhabditis elegans (invertebrata) / Tissue: Embryo

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.037 kPa
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.5 / Focused ion beam - Duration: 600 / Focused ion beam - Temperature: 95 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is FEI Aquilos cryo-FIB. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 120.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: Warp (ver. 1.0.7) / Number images used: 61

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