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- EMDB-14099: STRUCTURE OF THE HUMAN INNER KINETOCHORE CCAN COMPLEX AT 10A RESO... -

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Basic information

Entry
Database: EMDB / ID: EMD-14099
TitleSTRUCTURE OF THE HUMAN INNER KINETOCHORE CCAN COMPLEX AT 10A RESOLUTION
Map datamain map
Sample
  • Complex: human inner kinetochore CCAN complex
KeywordsINNER KINETOCHORE / CCAN / COMPLEX / DNA BINDING PROTEIN / CELL CYCLE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsVetter IR / Pesenti M / Raisch T
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2022
Title: Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization.
Authors: Marion E Pesenti / Tobias Raisch / Duccio Conti / Kai Walstein / Ingrid Hoffmann / Dorothee Vogt / Daniel Prumbaum / Ingrid R Vetter / Stefan Raunser / Andrea Musacchio /
Abstract: Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated ...Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.
History
DepositionDec 24, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14099.png

Downloads & links

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Map

FileDownload / File: emd_14099.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
2.72 Å/pix.
x 128 pix.
= 348.16 Å		2.72 Å/pix.
x 128 pix.
= 348.16 Å		2.72 Å/pix.
x 128 pix.
= 348.16 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.00036932708 - 0.60976714
Average (Standard dev.)0.0012277077 (±0.013529047)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 348.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14099_msk_1.map
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Mask #2

Fileemd_14099_msk_2.map
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Histogram (log scale)

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Mask #3

Fileemd_14099_msk_3.map
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Sample components

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Entire : human inner kinetochore CCAN complex

EntireName: human inner kinetochore CCAN complex
Components
  • Complex: human inner kinetochore CCAN complex

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Supramolecule #1: human inner kinetochore CCAN complex

SupramoleculeName: human inner kinetochore CCAN complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#15
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 451 KDa

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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

Concentration1.5 mg/mL
BufferpH: 6.8 / Details: 0.0025% Triton
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Spherical aberration corrector: Krios with CS corrector / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 14 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.8 e/Å2
Details: 2678 images were collected in movie-mode with 60 frames per image on Gatan K3 in superresolution mode with 0.34 A pixel size / native pixel size 0.68A
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 233598
Startup modelType of model: INSILICO MODEL / In silico model: Alphafold2 predictions
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 44216
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.1.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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