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- EMDB-4466: Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4466
TitleCryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box class
Map data
SampleCryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box class
  • (Anaphase-promoting complex subunit ...) x 10
  • Apc1
  • (Cell division cycle protein ...Cell cycle) x 4
  • Cyclin-A2
Function / homology
Function and homology information


positive regulation of anaphase-promoting complex-dependent catabolic process / regulation of mitotic cell cycle spindle assembly checkpoint / regulation of meiotic nuclear division / ubiquitin ligase activator activity / metaphase/anaphase transition of mitotic cell cycle / positive regulation of synapse maturation / positive regulation of mitotic metaphase/anaphase transition / anaphase-promoting complex / cellular response to luteinizing hormone stimulus / cellular response to cocaine ...positive regulation of anaphase-promoting complex-dependent catabolic process / regulation of mitotic cell cycle spindle assembly checkpoint / regulation of meiotic nuclear division / ubiquitin ligase activator activity / metaphase/anaphase transition of mitotic cell cycle / positive regulation of synapse maturation / positive regulation of mitotic metaphase/anaphase transition / anaphase-promoting complex / cellular response to luteinizing hormone stimulus / cellular response to cocaine / anaphase-promoting complex binding / regulation of exit from mitosis / regulation of mitotic metaphase/anaphase transition / cell cycle G1/S phase transition / protein K11-linked ubiquitination / regulation of dendrite development / ubiquitin-ubiquitin ligase activity / male pronucleus / mitotic cell cycle phase transition / female pronucleus / positive regulation of synaptic plasticity / cullin-RING ubiquitin ligase complex / cellular response to insulin-like growth factor stimulus / positive regulation of ubiquitin protein ligase activity / mitotic sister chromatid cohesion / cellular response to leptin stimulus / response to glucagon / mitotic metaphase plate congression / positive regulation of dendrite morphogenesis / cyclin-dependent protein serine/threonine kinase regulator activity / cullin family protein binding / cochlea development / cellular response to platelet-derived growth factor stimulus / mitotic spindle assembly checkpoint / positive regulation of axon extension / condensed chromosome kinetochore / regulation of DNA replication / intracellular anatomical structure / mitotic spindle assembly / cyclin A2-CDK2 complex / regulation of cyclin-dependent protein serine/threonine kinase activity / histone phosphorylation / regulation of mitotic cell cycle phase transition / molecular adaptor activity / nuclear periphery / cyclin-dependent protein kinase holoenzyme complex / cellular response to nitric oxide / regulation of mitotic cell cycle / kinetochore / cellular response to estradiol stimulus / animal organ regeneration / spindle / ubiquitin protein ligase activity / positive regulation of fibroblast proliferation / spindle pole / ubiquitin-protein transferase activity / histone deacetylase binding / anaphase-promoting complex-dependent catabolic process / cellular response to hypoxia / G2/M transition of mitotic cell cycle / mitotic cell cycle / Ras protein signal transduction / ubiquitin-dependent protein catabolic process / protein C-terminus binding / protein phosphatase binding / protein ubiquitination / protein deubiquitination / cell cycle / negative regulation of gene expression / cell division / centrosome / protein domain specific binding / nucleolus / ubiquitin protein ligase binding / positive regulation of cell population proliferation / viral process / protein kinase binding / positive regulation of transcription, DNA-templated / perinuclear region of cytoplasm / host cell nucleus / enzyme binding / protein-containing complex / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm
WD40/YVTN repeat-like-containing domain superfamily / Anaphase-promoting complex, subunit CDC26 / Cyclin, C-terminal domain / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 4 long domain / Anaphase-promoting complex subunit 4 / WD40 repeat, conserved site / APC10/DOC domain / Tetratricopeptide repeat ...WD40/YVTN repeat-like-containing domain superfamily / Anaphase-promoting complex, subunit CDC26 / Cyclin, C-terminal domain / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 4 long domain / Anaphase-promoting complex subunit 4 / WD40 repeat, conserved site / APC10/DOC domain / Tetratricopeptide repeat / Cyclin, N-terminal / WD40-repeat-containing domain / Galactose-binding-like domain superfamily / Anaphase-promoting complex subunit 4, metazoa / Anaphase-promoting complex subunit APC10/Doc1 / Cullin homology domain / Anaphase-promoting complex subunit 2, C-terminal / Cdc23 / Cyclin-like / Zinc finger, RING/FYVE/PHD-type / Apc13 / Tetratricopeptide repeat-containing domain / Zinc finger, RING-type / Tetratricopeptide-like helical domain superfamily / WD40 repeat / WD40-repeat-containing domain superfamily / Anaphase-promoting complex subunit 1, C-terminal / Cyclin / Anaphase-promoting complex subunit 5 / Cyclin-like superfamily / Winged helix DNA-binding domain superfamily / Tetratricopeptide repeat 1 / Winged helix-like DNA-binding domain superfamily / Cullin homology domain superfamily / The WD repeat Cdc20/Fizzy family / Cyclin-A, N-terminal / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit 15 / Cullin, N-terminal / Anaphase-promoting complex subunit 5 domain / Anaphase-promoting complex subunit 11
Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 2 / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 7 / Cell division cycle protein 23 homolog / Anaphase-promoting complex subunit 11 / Cell division cycle protein 16 homolog / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 16 ...Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 2 / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 7 / Cell division cycle protein 23 homolog / Anaphase-promoting complex subunit 11 / Cell division cycle protein 16 homolog / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit CDC26 / Cell division cycle protein 20 homolog / Anaphase-promoting complex subunit 15 / Cell division cycle protein 27 homolog / Cyclin-A2 / Anaphase-promoting complex subunit 10
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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.
Validation ReportPDB-ID: 6q6h

SummaryFull reportAbout validation report
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: 0.008
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6q6h
  • Surface level: 0.008
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4466.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.)
1.05 Å/pix.
x 400 pix.
= 418.8 Å
1.05 Å/pix.
x 400 pix.
= 418.8 Å
1.05 Å/pix.
x 400 pix.
= 418.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.047 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.034017798 - 0.05686256
Average (Standard dev.)0.0000014059206 (±0.003553726)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 418.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z418.800418.800418.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0160.042-0.000

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Supplemental data

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Additional map: sharpened map with B factor -30, lowpass filtered to 4A

Fileemd_4466_additional.map
Annotationsharpened map with B factor -30, lowpass filtered to 4A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: halfmap1

Fileemd_4466_half_map_1.map
Annotationhalfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: halfmap2

Fileemd_4466_half_map_2.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, ...

EntireName: Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box class
Number of components: 19

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Component #1: protein, Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 ...

ProteinName: Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box class
Recombinant expression: No
MassTheoretical: 1.3 MDa

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Component #2: protein, Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 ...

ProteinName: Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box class
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #3: protein, Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 ...

ProteinName: Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box class
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Esacherichia coli

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Component #4: protein, Anaphase-promoting complex subunit 10

ProteinName: Anaphase-promoting complex subunit 10 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 21.282143 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #5: protein, Anaphase-promoting complex subunit 15

ProteinName: Anaphase-promoting complex subunit 15 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 14.286727 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #6: protein, Apc1

ProteinName: Apc1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 207.240234 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #7: protein, Anaphase-promoting complex subunit 2

ProteinName: Anaphase-promoting complex subunit 2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 93.938977 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #8: protein, Anaphase-promoting complex subunit 4

ProteinName: Anaphase-promoting complex subunit 4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 92.219227 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #9: protein, Anaphase-promoting complex subunit 5

ProteinName: Anaphase-promoting complex subunit 5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 85.179766 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #10: protein, Cell division cycle protein 16 homolog

ProteinName: Cell division cycle protein 16 homologCell cycle / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 71.747516 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #11: protein, Anaphase-promoting complex subunit 11

ProteinName: Anaphase-promoting complex subunit 11 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.854647 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #12: protein, Anaphase-promoting complex subunit CDC26

ProteinName: Anaphase-promoting complex subunit CDC26 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 9.793999 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #13: protein, Anaphase-promoting complex subunit 13

ProteinName: Anaphase-promoting complex subunit 13 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.528309 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #14: protein, Anaphase-promoting complex subunit 16

ProteinName: Anaphase-promoting complex subunit 16 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.677995 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #15: protein, Cell division cycle protein 27 homolog

ProteinName: Cell division cycle protein 27 homologCell cycle / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 91.973125 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #16: protein, Anaphase-promoting complex subunit 7

ProteinName: Anaphase-promoting complex subunit 7 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 66.929367 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #17: protein, Cell division cycle protein 23 homolog

ProteinName: Cell division cycle protein 23 homologCell cycle / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 68.921031 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #18: protein, Cell division cycle protein 20 homolog

ProteinName: Cell division cycle protein 20 homologCell cycle / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 54.796508 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: unidentified baculovirus

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Component #19: protein, Cyclin-A2

ProteinName: Cyclin-A2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 44.427766 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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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: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
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: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 117044
3D reconstructionSoftware: RELION / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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