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- EMDB-0052: Masked Core Centromere Binding Factor 3 (CBF3) with monomeric Ndc10 -

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Basic information

Entry
Database: EMDB / ID: EMD-0052
TitleMasked Core Centromere Binding Factor 3 (CBF3) with monomeric Ndc10
Map datafocused refinement of Ndc10D1-2 in CBF3
Sample
  • Complex: Core CBF3 in complex with Ndc10 D1-2
    • Complex: Cep3 homodimer
    • Complex: Skp1S-phase kinase-associated protein 1
    • Complex: Ctf13
    • Complex: Ndc10 comprising domains 1-2
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsZhang WJ / Lukoynova N
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.
History
DepositionJun 13, 2018-
Header (metadata) releaseJul 4, 2018-
Map releaseAug 1, 2018-
UpdateNov 28, 2018-
Current statusNov 28, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0221
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0221
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0052.png

Downloads & links

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Map

FileDownload / File: emd_0052.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfocused refinement of Ndc10D1-2 in CBF3
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0221 / Movie #1: 0.0221
Minimum - Maximum-0.034055203 - 0.092103794
Average (Standard dev.)0.0005317477 (±0.0039967815)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0340.0920.001

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Supplemental data

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Sample components

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Entire : Core CBF3 in complex with Ndc10 D1-2

EntireName: Core CBF3 in complex with Ndc10 D1-2
Components
  • Complex: Core CBF3 in complex with Ndc10 D1-2
    • Complex: Cep3 homodimer
    • Complex: Skp1S-phase kinase-associated protein 1
    • Complex: Ctf13
    • Complex: Ndc10 comprising domains 1-2

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Supramolecule #1: Core CBF3 in complex with Ndc10 D1-2

SupramoleculeName: Core CBF3 in complex with Ndc10 D1-2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: The truncated CBF3 complex, recombinantly expressed in Saccharomyces cerevisiae. It comprises a Cep3 homodimer, in which the binuclear zinc cluster domains are truncated, full length ...Details: The truncated CBF3 complex, recombinantly expressed in Saccharomyces cerevisiae. It comprises a Cep3 homodimer, in which the binuclear zinc cluster domains are truncated, full length heterodimer of Skp1 and Ctf13, and a monomeric construct Ndc10 comprising domains 1-2.
Molecular weightTheoretical: 286 KDa

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Supramolecule #2: Cep3 homodimer

SupramoleculeName: Cep3 homodimer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: Skp1

SupramoleculeName: Skp1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: modified pRS424 and pRS426

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Supramolecule #4: Ctf13

SupramoleculeName: Ctf13 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #5: Ndc10 comprising domains 1-2

SupramoleculeName: Ndc10 comprising domains 1-2 / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: 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

Concentration0.15 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
15.0 mMTris base
2.0 mMDTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
DetailsThe sample is homogeneous and well-dispersed on grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47170 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 2003 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 214608
Details: Number of particles after selection by 2D classification
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Software - details: CTF parameters were estimated using CTFFIND4
Startup modelType of model: OTHER
Details: Cryosparc was used to generate a startup model from a smaller dataset collected in-house. This was then low pass filtered to 60 Angstroms
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0)
Details: This map was generated using focused refinement of the Ndc10 component of the map.
Number images used: 22965
DetailsThe selected images were high-pass filtered and normalized.
FSC plot (resolution estimation)

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Atomic model buiding 1

Details"Fit in map" function used to place 6FE8 and 4ACO with out further refinement or model building

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