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- EMDB-44864: Kinetochore on isolated human mitotic chromosome under conditions... -

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Basic information

Entry
Database: EMDB / ID: EMD-44864
TitleKinetochore on isolated human mitotic chromosome under conditions of prolonged spindle detachment
Map dataKinetochore on isolated human mitotic chromosome under conditions of prolonged spindle detachment
Sample
  • Organelle or cellular component: Mitotic chromosome
KeywordsKinetochore / Chromosome / CELL CYCLE
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsKixmoeller K / Chang YW / Black BE
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)261198 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)134020 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)130302 United States
David and Lucile Packard Foundation2019-69645 United States
Burroughs Wellcome Fund1022785 United States
CitationJournal: Cell / Year: 2025
Title: Centromeric chromatin clearings demarcate the site of kinetochore formation.
Authors: Kathryn Kixmoeller / Ekaterina V Tarasovetc / Elie Mer / Yi-Wei Chang / Ben E Black /
Abstract: The centromere is the chromosomal locus that recruits the kinetochore, directing faithful propagation of the genome during cell division. Using cryo-ET on human mitotic chromosomes, we reveal a ...The centromere is the chromosomal locus that recruits the kinetochore, directing faithful propagation of the genome during cell division. Using cryo-ET on human mitotic chromosomes, we reveal a distinctive architecture at the centromere: clustered 20- to 25-nm nucleosome-associated complexes within chromatin clearings that delineate them from surrounding chromatin. Centromere components CENP-C and CENP-N are each required for the integrity of the complexes, while CENP-C is also required to maintain the chromatin clearing. We find that CENP-C is required in mitosis, not just for kinetochore assembly, likely reflecting its role in organizing the inner kinetochore during chromosome segregation. We further visualize the scaffold of the fibrous corona, a structure amplified at unattached kinetochores, revealing crescent-shaped parallel arrays of fibrils extending >1 μm. Thus, we reveal how the organization of centromeric chromatin creates a clearing at the site of kinetochore formation as well as the nature of kinetochore amplification mediated by corona fibrils.
History
DepositionMay 13, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44864.png

Downloads & links

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Map

FileDownload / File: emd_44864.map.gz / Format: CCP4 / Size: 569 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationKinetochore on isolated human mitotic chromosome under conditions of prolonged spindle detachment
Voxel sizeX=Y=Z: 8.551 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)35.106119999999997 (±22.185064000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-209209-203
Dimensions14401023405
Spacing10231440405
CellA: 8747.673 Å / B: 12313.439 Å / C: 3463.1548 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Mitotic chromosome

EntireName: Mitotic chromosome
Components
  • Organelle or cellular component: Mitotic chromosome

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Supramolecule #1: Mitotic chromosome

SupramoleculeName: Mitotic chromosome / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Centromere region of mitotic chromosome including the kinetochore after prolonged spindle detachment
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: LEICA EM GP
DetailsKinetochore on isolated human mitotic chromosome under conditions of prolonged spindle detachment
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Ted Pella / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.13 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 56

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