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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: 0052
TitleMasked Core Centromere Binding Factor 3 (CBF3) with monomeric Ndc10
Map datafocused refinement of Ndc10D1-2 in CBF3
SampleCore CBF3 in complex with Ndc10 D1-2:
Cep3 homodimer / Skp1 / Ctf13 / Ndc10 comprising domains 1-2
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 4.4 Å resolution
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 Lukoynova / 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.
DateDeposition: Jun 13, 2018 / Header (metadata) release: Jul 4, 2018 / Map release: Aug 1, 2018 / Last update: Aug 29, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0221
  • Imaged by UCSF Chimera
  • Download
  • 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

Downloads & links

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Map

Fileemd_0052.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.06 Å/pix.
= 271.36 Å
256 pix
1.06 Å/pix.
= 271.36 Å
256 pix
1.06 Å/pix.
= 271.36 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 Level:0.0221 (by author), 0.0221 (movie #1):
Minimum - Maximum-0.034055203 - 0.092103794
Average (Standard dev.)0.0005317477 (0.0039967815)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.0.0.
Limit255.255.255.
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
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
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.
Number of components: 5

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Component #1: protein, Core CBF3 in complex with Ndc10 D1-2

ProteinName: Core CBF3 in complex with Ndc10 D1-2
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.
Recombinant expression: No
MassTheoretical: 286 kDa

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Component #2: protein, Cep3 homodimer

ProteinName: Cep3 homodimer / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, Skp1

ProteinName: Skp1 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast) / Vector: modified pRS424 and pRS426

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Component #4: protein, Ctf13

ProteinName: Ctf13 / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #5: protein, Ndc10 comprising domains 1-2

ProteinName: Ndc10 comprising domains 1-2 / Recombinant expression: No
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.: 0.15 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: 5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 47170. 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 acquisitionNumber of digital images: 2003

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 22965
Details: The selected images were high-pass filtered and normalized.
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF
Details: This map was generated using focused refinement of the Ndc10 component of the map.
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Details: "Fit in map" function used to place 6FE8 and 4ACO with out further refinement or model building

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