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- EMDB-17371: Multibody map of CCAN(Topo) bound to CON3 DNA as part of the Inne... -

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Basic information

Entry
Database: EMDB / ID: EMD-17371
TitleMultibody map of CCAN(Topo) bound to CON3 DNA as part of the Inner kinetochore.
Map data
Sample
  • Complex: A complex of CBF1-CCAN bound to centromeric C0N3 DNA
Keywordskinetochore / point centromere / CENP-A nucleosome / topological entrapment / centromeric DNA / CELL CYCLE
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDendooven TD / Zhang Z / Yang J / McLaughlin S / Schwabb J / Scheres S / Yatskevich S / Barford D
Funding support United Kingdom, Germany, 4 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
UK Research and Innovation (UKRI)MC_UP_ A025_1013 United Kingdom
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere.
Authors: Tom Dendooven / Ziguo Zhang / Jing Yang / Stephen H McLaughlin / Johannes Schwab / Sjors H W Scheres / Stanislau Yatskevich / David Barford /
Abstract: The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) ...The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-A). This revealed a central CENP-A with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-A can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.
History
DepositionMay 16, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17371.png

Downloads & links

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Map

FileDownload / File: emd_17371.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 352 pix.
= 380.16 Å		1.08 Å/pix.
x 352 pix.
= 380.16 Å		1.08 Å/pix.
x 352 pix.
= 380.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0094
Minimum - Maximum-0.034672912 - 0.06235313
Average (Standard dev.)-0.000009692292 (±0.0018706025)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 380.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_17371_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17371_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : A complex of CBF1-CCAN bound to centromeric C0N3 DNA

EntireName: A complex of CBF1-CCAN bound to centromeric C0N3 DNA
Components
  • Complex: A complex of CBF1-CCAN bound to centromeric C0N3 DNA

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Supramolecule #1: A complex of CBF1-CCAN bound to centromeric C0N3 DNA

SupramoleculeName: A complex of CBF1-CCAN bound to centromeric C0N3 DNA / type: complex / ID: 1 / Parent: 0
Macromolecule list: #1-#5, #7, #9, #8, #10-#11, #6, #13-#15, #12, #16-#23
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 108672
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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