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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


Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / RHO GTPases Activate Formins / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Regulation of APC/C activators between G1/S and early anaphase / Phosphorylation of the APC/C / Phosphorylation of Emi1 / APC-Cdc20 mediated degradation of Nek2A / SCF(Skp2)-mediated degradation of p27/p21 ...Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / RHO GTPases Activate Formins / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Regulation of APC/C activators between G1/S and early anaphase / Phosphorylation of the APC/C / Phosphorylation of Emi1 / APC-Cdc20 mediated degradation of Nek2A / SCF(Skp2)-mediated degradation of p27/p21 / Separation of Sister Chromatids / Resolution of Sister Chromatid Cohesion / Senescence-Associated Secretory Phenotype (SASP) / DNA Damage/Telomere Stress Induced Senescence / Ub-specific processing proteases / APC/C:Cdc20 mediated degradation of Securin / Processing of DNA double-strand break ends / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / Regulation of TP53 Activity through Phosphorylation / Regulation of TP53 Degradation / Mitotic Prometaphase / G2 Phase / Orc1 removal from chromatin / CDK-mediated phosphorylation and removal of Cdc6 / Cyclin A/B1/B2 associated events during G2/M transition / p53-Dependent G1 DNA Damage Response / Cyclin A:Cdk2-associated events at S phase entry / Antigen processing: Ubiquitination & Proteasome degradation / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / APC/C:Cdc20 mediated degradation of mitotic proteins / SCF-beta-TrCP mediated degradation of Emi1 / APC/C:Cdc20 mediated degradation of Cyclin B / Inactivation of APC/C via direct inhibition of the APC/C complex / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / G0 and Early G1 / Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes / Autodegradation of Cdh1 by Cdh1:APC/C / regulation of mitotic cell cycle spindle assembly checkpoint / metaphase/anaphase transition of mitotic cell cycle / regulation of meiotic nuclear division / positive regulation of synapse maturation / ubiquitin-protein transferase activator activity / positive regulation of synaptic plasticity / positive regulation of mitotic metaphase/anaphase transition / anaphase-promoting complex / cullin-RING ubiquitin ligase 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 / ubiquitin-ubiquitin ligase activity / positive regulation of ubiquitin protein ligase activity / regulation of dendrite development / mitotic sister chromatid cohesion / mitotic cell cycle phase transition / male pronucleus / protein K11-linked ubiquitination / female pronucleus / cellular response to insulin-like growth factor stimulus / mitotic metaphase plate congression / cellular response to leptin stimulus / response to glucagon / cullin family protein binding / positive regulation of dendrite morphogenesis / cyclin-dependent protein serine/threonine kinase regulator activity / cochlea development / regulation of mitotic cell cycle / cellular response to platelet-derived growth factor stimulus / intracellular / positive regulation of axon extension / condensed chromosome kinetochore / mitotic spindle assembly checkpoint / regulation of DNA replication / mitotic spindle assembly / regulation of cyclin-dependent protein serine/threonine kinase activity / cyclin A2-CDK2 complex / histone phosphorylation / regulation of mitotic cell cycle phase transition / nuclear periphery / cyclin-dependent protein kinase holoenzyme complex / cellular response to nitric oxide / kinetochore / cellular response to estradiol stimulus / animal organ regeneration / spindle / ubiquitin protein ligase activity / spindle pole / positive regulation of fibroblast proliferation / ubiquitin-protein transferase activity / histone deacetylase binding / cellular response to hypoxia / anaphase-promoting complex-dependent catabolic process / mitotic cell cycle / Ras protein signal transduction / G2/M transition of mitotic cell cycle / ubiquitin-dependent protein catabolic process / protein phosphatase binding / protein C-terminus binding
Anaphase-promoting complex subunit APC10/Doc1 / Tetratricopeptide repeat-containing domain / Anaphase-promoting complex subunit 4 long domain / Anaphase-promoting complex subunit 4 / WD40 repeat, conserved site / Tetratricopeptide repeat / Anaphase-promoting complex, subunit CDC26 / WD40-repeat-containing domain / Anaphase-promoting complex subunit 4, metazoa / Cullin family profile. ...Anaphase-promoting complex subunit APC10/Doc1 / Tetratricopeptide repeat-containing domain / Anaphase-promoting complex subunit 4 long domain / Anaphase-promoting complex subunit 4 / WD40 repeat, conserved site / Tetratricopeptide repeat / Anaphase-promoting complex, subunit CDC26 / WD40-repeat-containing domain / Anaphase-promoting complex subunit 4, metazoa / Cullin family profile. / Cullin homology domain / WD40/YVTN repeat-like-containing domain superfamily / Anaphase-promoting complex subunit 2, C-terminal / Cyclin-like / Zinc finger, RING/FYVE/PHD-type / Tetratricopeptide-like helical domain superfamily / Anaphase-promoting complex subunit 11 / Galactose-binding-like domain superfamily / Apc13 / Cdc23 / Cyclin, N-terminal / APC10/DOC domain / Cyclin, C-terminal domain / Zinc finger, RING-type / WD40 repeat / Tetratricopeptide repeat 1 / Cullin, N-terminal / Trp-Asp (WD) repeats profile. / Zinc finger RING-type profile. / TPR repeat region circular profile. / TPR repeat profile. / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 5 domain / Anaphase-promoting complex, subunit 10 (APC10) / Cyclins signature. / Anaphase-promoting complex, subunit 16 / N-terminal region of cyclin_N / Anaphase-promoting complex subunit 15 / Tetratricopeptide repeat / Anaphase-promoting complex, cyclosome, subunit 4 / Anaphase-promoting complex subunit 4 WD40 domain / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 11 RING-H2 finger / Anaphase-promoting complex APC subunit CDC26 / Anaphase promoting complex (APC) subunit 2 / Apc13p protein / Anaphase promoting complex subunit 8 / Cdc23 / Cyclin, C-terminal domain / Anaphase-promoting complex subunit 15 / Cullin family / Tetratricopeptide repeat / WD domain, G-beta repeat / Cyclin, N-terminal domain / Cyclin / Anaphase-promoting complex subunit 5 / Cyclin-like superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / Cullin homology domain superfamily / The WD repeat Cdc20/Fizzy family / Cyclin-A, N-terminal / Anaphase-promoting complex subunit 16 / DOC domain profile. / Trp-Asp (WD) repeats circular profile. / Trp-Asp (WD) repeats signature.
Anaphase-promoting complex subunit 11 / 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 / Cell division cycle protein 16 homolog / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit CDC26 ...Anaphase-promoting complex subunit 11 / 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 / 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
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  • 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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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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