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- EMDB-4241: Cryo-EM structure of the core Centromere Binding Factor 3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-4241
TitleCryo-EM structure of the core Centromere Binding Factor 3 complex
Map data
SampleThe core Centromere Binding Factor 3 complex
  • (Centromere DNA-binding protein complex CBF3 subunit ...) x 2
  • Suppressor of kinetochore protein 1
Function / homology
Function and homology information


RAVE complex / CBF3 complex / septin ring assembly / negative regulation of cytoplasmic translation / centromeric DNA binding / protein neddylation / regulation of exit from mitosis / vacuolar acidification / kinetochore assembly / condensed nuclear chromosome kinetochore ...RAVE complex / CBF3 complex / septin ring assembly / negative regulation of cytoplasmic translation / centromeric DNA binding / protein neddylation / regulation of exit from mitosis / vacuolar acidification / kinetochore assembly / condensed nuclear chromosome kinetochore / SCF ubiquitin ligase complex / exit from mitosis / cullin family protein binding / DNA binding, bending / cellular protein-containing complex assembly / DNA replication origin binding / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / mitotic spindle assembly checkpoint / condensed chromosome kinetochore / regulation of protein-containing complex assembly / kinetochore / G1/S transition of mitotic cell cycle / mitotic cell cycle / G2/M transition of mitotic cell cycle / ubiquitin-dependent protein catabolic process / protein ubiquitination / DNA-binding transcription factor activity, RNA polymerase II-specific / host cell nucleus / zinc ion binding / identical protein binding / nucleus / cytoplasm
SKP1 component, POZ domain / S-phase kinase-associated protein 1 / Zn(2)-C6 fungal-type DNA-binding domain superfamily / Zn(2)-C6 fungal-type DNA-binding domain / S-phase kinase-associated protein 1-like / SKP1/BTB/POZ domain superfamily / SKP1-like, dimerisation domain superfamily / SKP1 component, dimerisation / Leucine-rich repeat domain superfamily / Centromere DNA-binding protein complex CBF3 subunit B, C-terminal
Centromere DNA-binding protein complex CBF3 subunit C / Centromere DNA-binding protein complex CBF3 subunit B / Suppressor of kinetochore protein 1
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZhang WJ / Lukoynova N / Miah S / Vaughan CK
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/J007595/1 United Kingdom
CitationJournal: Cell Rep / Year: 2018
Title: Insights into Centromere DNA Bending Revealed by the Cryo-EM Structure of the Core Centromere Binding Factor 3 with Ndc10.
Authors: Wenjuan Zhang / Natalya Lukoyanova / Shomon Miah / Jonathan Lucas / Cara K Vaughan /
Abstract: The centromere binding factor 3 (CBF3) complex binds the third centromere DNA element in organisms with point centromeres, such as S. cerevisiae. It is an essential complex for assembly of the ...The centromere binding factor 3 (CBF3) complex binds the third centromere DNA element in organisms with point centromeres, such as S. cerevisiae. It is an essential complex for assembly of the kinetochore in these organisms, as it facilitates genetic centromere specification and allows association of all other kinetochore components. We determined high-resolution structures of the core complex of CBF3 alone and in association with a monomeric construct of Ndc10, using cryoelectron microscopy (cryo-EM). We identify the DNA-binding site of the complex and present a model in which CBF3 induces a tight bend in centromeric DNA, thus facilitating assembly of the centromeric nucleosome.
Validation ReportPDB-ID: 6fe8

SummaryFull reportAbout validation report
History
DepositionDec 30, 2017-
Header (metadata) releaseJan 10, 2018-
Map releaseAug 1, 2018-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0664
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0664
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6fe8
  • Surface level: 0.0664
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4241.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 212. Å
1.06 Å/pix.
x 200 pix.
= 212. Å
1.06 Å/pix.
x 200 pix.
= 212. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0664 / Movie #1: 0.0664
Minimum - Maximum-0.18915181 - 0.3365617
Average (Standard dev.)0.00065497926 (±0.0142806135)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.000212.000212.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1890.3370.001

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Supplemental data

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Segmentation: #1

Fileemd_4241_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map of the final map, which was used for FSC calculation.

Fileemd_4241_half_map_1.map
AnnotationHalf map of the final map, which was used for FSC calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: The other half map of the final map,...

Fileemd_4241_half_map_2.map
AnnotationThe other half map of the final map, which was used for FSC calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire The core Centromere Binding Factor 3 complex

EntireName: The core Centromere Binding Factor 3 complex
Details: The core CBF3 complex, recombinantly expressed in Saccharomyces cerevisiae. It comprises a Cep3 homodimer, in which the binuclear zinc cluster domains are truncated, and full length Skp1 and Ctf13 components.
Number of components: 4

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Component #1: protein, The core Centromere Binding Factor 3 complex

ProteinName: The core Centromere Binding Factor 3 complex
Details: The core CBF3 complex, recombinantly expressed in Saccharomyces cerevisiae. It comprises a Cep3 homodimer, in which the binuclear zinc cluster domains are truncated, and full length Skp1 and Ctf13 components.
Recombinant expression: No
MassTheoretical: 220 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Centromere DNA-binding protein complex CBF3 subunit B

ProteinName: Centromere DNA-binding protein complex CBF3 subunit B / Details: Model is numbered according to / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 68.454125 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, Suppressor of kinetochore protein 1

ProteinName: Suppressor of kinetochore protein 1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 22.558451 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #4: protein, Centromere DNA-binding protein complex CBF3 subunit C

ProteinName: Centromere DNA-binding protein complex CBF3 subunit C / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 60.899961 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.12 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 4.06 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 47170.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisition #1Number of digital images: 1236
Image acquisition #2Number of digital images: 1101

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 69392
Details: The selected images were high-pass filtered and normalized.
3D reconstruction #1Algorithm: FOURIER SPACE / Software: RELION / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF
Details: 3D auto-refinement of all the good particles from 2D classification, provided a reconstruction at 4.7 angstrom overall resolution. After post-process by RELION and sharpened by a negative B- ...Details: 3D auto-refinement of all the good particles from 2D classification, provided a reconstruction at 4.7 angstrom overall resolution. After post-process by RELION and sharpened by a negative B-factor using an automated procedure resulting in a 4.1 angstrom reconstruction.
3D reconstruction #2Algorithm: FOURIER SPACE / Software: RELION / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF
Details: 187, 606 particles were in the best two 3D classes. 3D auto-refinement of the 3D joined classes against the corresponding particles resulted in a final reconstruction.
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Cross-correlation coefficient / Refinement space: REAL
Input PDB model: 2VEQ, 1NEX
Chain ID: A, A
Output model

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