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- EMDB-4463: Cryo-EM reconstruction of the APC/C-MCC-Cdk2-cyclinA2-Cks2 comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-4463
TitleCryo-EM reconstruction of the APC/C-MCC-Cdk2-cyclinA2-Cks2 complex in the open conformation
Map data
SampleCryo-EM reconstruction of the APC/C-MCC-Cdk2-cyclinA2-Cks2 complex in the open conformation:
Apc1 / Apc2 / Apc3 / Apc4 / Apc5 / Apc6 / Apc7 / Apc8 / Apc10 / Apc11 ...Apc1 / Apc2 / Apc3 / Apc4 / Apc5 / Apc6 / Apc7 / Apc8 / Apc10 / Apc11 / Apc12 / Apc13 / Apc15 / Apc16 / Cdc20 / Cdk2Cyclin-dependent kinase 2 / Cyclin A2 / Cks2 / BubR1BUB1B / Mad2 / Bub3
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhang S / Barford D
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1021/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
CitationJournal: Nat Commun / Year: 2019
Title: Cyclin A2 degradation during the spindle assembly checkpoint requires multiple binding modes to the APC/C.
Authors: Suyang Zhang / Thomas Tischer / David Barford /
Abstract: The anaphase-promoting complex/cyclosome (APC/C) orchestrates cell cycle progression by controlling the temporal degradation of specific cell cycle regulators. Although cyclin A2 and cyclin B1 are ...The anaphase-promoting complex/cyclosome (APC/C) orchestrates cell cycle progression by controlling the temporal degradation of specific cell cycle regulators. Although cyclin A2 and cyclin B1 are both targeted for degradation by the APC/C, during the spindle assembly checkpoint (SAC), the mitotic checkpoint complex (MCC) represses APC/C's activity towards cyclin B1, but not cyclin A2. Through structural, biochemical and in vivo analysis, we identify a non-canonical D box (D2) that is critical for cyclin A2 ubiquitination in vitro and degradation in vivo. During the SAC, cyclin A2 is ubiquitinated by the repressed APC/C-MCC, mediated by the cooperative engagement of its KEN and D2 boxes, ABBA motif, and the cofactor Cks. Once the SAC is satisfied, cyclin A2 binds APC/C-Cdc20 through two mutually exclusive binding modes, resulting in differential ubiquitination efficiency. Our findings reveal that a single substrate can engage an E3 ligase through multiple binding modes, affecting its degradation timing and efficiency.
History
DepositionDec 11, 2018-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 2019-
UpdateSep 11, 2019-
Current statusSep 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4463.map.gz / Format: CCP4 / Size: 244.1 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 400 pix.
= 424. Å
1.06 Å/pix.
x 400 pix.
= 424. Å
1.06 Å/pix.
x 400 pix.
= 424. 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: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.017014023 - 0.06736204
Average (Standard dev.)0.00013174274 (±0.0034849634)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 423.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z424.000424.000424.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0660.1340.000

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Supplemental data

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Sample components

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Entire Cryo-EM reconstruction of the APC/C-MCC-Cdk2-cyclinA2-Cks2 comple...

EntireName: Cryo-EM reconstruction of the APC/C-MCC-Cdk2-cyclinA2-Cks2 complex in the open conformation
Number of components: 22

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Component #1: protein, Cryo-EM reconstruction of the APC/C-MCC-Cdk2-cyclinA2-Ck...

ProteinName: Cryo-EM reconstruction of the APC/C-MCC-Cdk2-cyclinA2-Cks2 complex in the open conformation
Recombinant expression: No
MassTheoretical: 1.6 MDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #2: protein, Apc1

ProteinName: Apc1 / Recombinant expression: No

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Component #3: protein, Apc2

ProteinName: Apc2 / Recombinant expression: No

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Component #4: protein, Apc3

ProteinName: Apc3 / Recombinant expression: No

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Component #5: protein, Apc4

ProteinName: Apc4 / Recombinant expression: No

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Component #6: protein, Apc5

ProteinName: Apc5 / Recombinant expression: No

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Component #7: protein, Apc6

ProteinName: Apc6 / Recombinant expression: No

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Component #8: protein, Apc7

ProteinName: Apc7 / Recombinant expression: No

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Component #9: protein, Apc8

ProteinName: Apc8 / Recombinant expression: No

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Component #10: protein, Apc10

ProteinName: Apc10 / Recombinant expression: No

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Component #11: protein, Apc11

ProteinName: Apc11 / Recombinant expression: No

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Component #12: protein, Apc12

ProteinName: Apc12 / Recombinant expression: No

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Component #13: protein, Apc13

ProteinName: Apc13 / Recombinant expression: No

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Component #14: protein, Apc15

ProteinName: Apc15 / Recombinant expression: No

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Component #15: protein, Apc16

ProteinName: Apc16 / Recombinant expression: No

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Component #16: protein, Cdc20

ProteinName: Cdc20 / Recombinant expression: No

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Component #17: protein, Cdk2

ProteinName: Cdk2Cyclin-dependent kinase 2 / Recombinant expression: No

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Component #18: protein, Cyclin A2

ProteinName: Cyclin A2 / Recombinant expression: No

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Component #19: protein, Cks2

ProteinName: Cks2 / Recombinant expression: No

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Component #20: protein, BubR1

ProteinName: BubR1BUB1B / Recombinant expression: No

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Component #21: protein, Mad2

ProteinName: Mad2 / Recombinant expression: No

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Component #22: protein, Bub3

ProteinName: Bub3 / Recombinant expression: No

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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 / Buffer solution: 20mM Hepes, 150mM NaCl, 0.5mM TCEP / pH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 28 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 184058
3D reconstructionSoftware: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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