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- PDB-6q6h: Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, ... -

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Entry
Database: PDB / ID: 6q6h
TitleCryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box class
Components
  • (Anaphase-promoting complex subunit ...) x 10
  • (Cell division cycle protein ...Cell cycle) x 4
  • Apc1
  • Cyclin-A2
KeywordsCELL CYCLE / spindle assembly checkpoint / anaphase-promoting complex / cyclin / ubiquitination
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
Zinc finger, RING-type / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 4 long domain / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 11 / Anaphase-promoting complex subunit 5 domain / Anaphase-promoting complex subunit 15 / Cyclin-A, N-terminal ...Zinc finger, RING-type / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 4 long domain / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 11 / Anaphase-promoting complex subunit 5 domain / Anaphase-promoting complex subunit 15 / Cyclin-A, N-terminal / Tetratricopeptide repeat / The WD repeat Cdc20/Fizzy family / Cullin homology domain superfamily / WD40-repeat-containing domain / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / Winged helix DNA-binding domain superfamily / Cyclin-like superfamily / WD40 repeat, conserved site / Cullin, N-terminal / Cyclin / Tetratricopeptide-like helical domain superfamily / Anaphase-promoting complex subunit APC10/Doc1 / 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 repeat-containing domain / Galactose-binding-like domain superfamily / Tetratricopeptide repeat 1 / Apc13 / Cdc23 / Cyclin, N-terminal / APC10/DOC domain / Cyclin, C-terminal domain / Anaphase-promoting complex, subunit CDC26 / WD40 repeat / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 4, metazoa / Anaphase-promoting complex subunit 1, C-terminal / Anaphase-promoting complex subunit 1 / Tetratricopeptide repeat / Anaphase-promoting complex, cyclosome, subunit 3 / Tetratricopeptide repeat / Anaphase-promoting complex, cyclosome, subunit 4 / Anaphase-promoting complex, subunit 10 (APC10) / Apc13p protein / Cullin family / Anaphase promoting complex (APC) subunit 2 / Anaphase-promoting complex subunit 4 WD40 domain / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex, subunit 16 / WD domain, G-beta repeat / Cyclin, N-terminal domain / Cyclin, C-terminal domain / N-terminal region of cyclin_N / Anaphase-promoting complex APC subunit CDC26 / Anaphase promoting complex subunit 8 / Cdc23 / Anaphase-promoting complex subunit 15 / Tetratricopeptide repeat / Anaphase-promoting complex subunit 11 RING-H2 finger / Anaphase-promoting complex sub unit 1 C-terminal domain / Tetratricopeptide repeat / Tetratricopeptide repeat / Tetratricopeptide repeat domain / Zinc/RING finger domain, C3HC4 (zinc finger) / Herpes Virus-1 / Galactose-binding domain-like / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / Jelly Rolls / Sandwich / 2-Layer Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Cell division cycle protein 20 homolog / Cell division cycle protein 16 homolog / Cyclin-A2 / Cell division cycle protein 27 homolog / Anaphase-promoting complex subunit 15 / 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 20 homolog / Cell division cycle protein 16 homolog / Cyclin-A2 / Cell division cycle protein 27 homolog / Anaphase-promoting complex subunit 15 / 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 / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit CDC26 / Anaphase-promoting complex subunit 10
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang, S. / Barford, D.
Funding support United Kingdom, 2items
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 Report
SummaryFull reportAbout validation report
History
DepositionDec 11, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
L: Anaphase-promoting complex subunit 10
D: Anaphase-promoting complex subunit 15
A: Apc1
N: Anaphase-promoting complex subunit 2
I: Anaphase-promoting complex subunit 4
O: Anaphase-promoting complex subunit 5
K: Cell division cycle protein 16 homolog
C: Anaphase-promoting complex subunit 11
G: Anaphase-promoting complex subunit CDC26
W: Anaphase-promoting complex subunit CDC26
M: Anaphase-promoting complex subunit 13
H: Anaphase-promoting complex subunit 16
J: Cell division cycle protein 27 homolog
P: Cell division cycle protein 27 homolog
Q: Cell division cycle protein 16 homolog
Y: Anaphase-promoting complex subunit 7
U: Cell division cycle protein 23 homolog
V: Cell division cycle protein 23 homolog
Z: Anaphase-promoting complex subunit 7
R: Cell division cycle protein 20 homolog
S: Cyclin-A2


Theoretical massNumber of molelcules
Total (without water)1,262,16221
Polymers1,262,16221
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area112190 Å2
ΔGint-554 kcal/mol
Surface area333360 Å2
MethodPISA

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Components

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Anaphase-promoting complex subunit ... , 10 types, 12 molecules LDNIOCGWMHYZ

#1: Protein Anaphase-promoting complex subunit 10 / / APC10 / Cyclosome subunit 10


Mass: 21282.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC10, APC10 / Production host: unidentified baculovirus / References: UniProt: Q9UM13
#2: Protein Anaphase-promoting complex subunit 15 / / APC15


Mass: 14286.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC15, C11orf51, HSPC020 / Production host: unidentified baculovirus / References: UniProt: P60006
#4: Protein Anaphase-promoting complex subunit 2 / / APC2 / Cyclosome subunit 2


Mass: 93938.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC2, APC2, KIAA1406 / Production host: unidentified baculovirus / References: UniProt: Q9UJX6
#5: Protein Anaphase-promoting complex subunit 4 / / APC4 / Cyclosome subunit 4


Mass: 92219.227 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC4, APC4 / Production host: unidentified baculovirus / References: UniProt: Q9UJX5
#6: Protein Anaphase-promoting complex subunit 5 / / APC5 / Cyclosome subunit 5


Mass: 85179.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC5, APC5 / Production host: unidentified baculovirus / References: UniProt: Q9UJX4
#8: Protein Anaphase-promoting complex subunit 11 / / APC11 / Cyclosome subunit 11 / Hepatocellular carcinoma-associated RING finger protein


Mass: 9854.647 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC11, HSPC214 / Production host: unidentified baculovirus / References: UniProt: Q9NYG5
#9: Protein Anaphase-promoting complex subunit CDC26 / / Anaphase-promoting complex subunit 12 / APC12 / Cell division cycle protein 26 homolog


Mass: 9793.999 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC26, ANAPC12, C9orf17 / Production host: unidentified baculovirus / References: UniProt: Q8NHZ8
#10: Protein Anaphase-promoting complex subunit 13 / / APC13 / Cyclosome subunit 13


Mass: 8528.309 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC13 / Production host: unidentified baculovirus / References: UniProt: Q9BS18
#11: Protein Anaphase-promoting complex subunit 16 / / APC16 / Cyclosome subunit 16


Mass: 11677.995 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC16, C10orf104, CENP-27 / Production host: unidentified baculovirus / References: UniProt: Q96DE5
#13: Protein Anaphase-promoting complex subunit 7 / / APC7 / Cyclosome subunit 7


Mass: 66929.367 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC7, APC7 / Production host: unidentified baculovirus / References: UniProt: Q9UJX3

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Protein , 2 types, 2 molecules AS

#3: Protein Apc1


Mass: 207240.234 Da / Num. of mol.: 1 / Mutation: deletion of residues 307-395
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: unidentified baculovirus / References: UniProt: Q9H1A4*PLUS
#16: Protein Cyclin-A2 / Cyclin-A / Cyclin A


Mass: 44427.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCNA2, CCN1, CCNA / Production host: Escherichia coli (E. coli) / References: UniProt: P20248

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Cell division cycle protein ... , 4 types, 7 molecules KQJPUVR

#7: Protein Cell division cycle protein 16 homolog / Cell cycle / Anaphase-promoting complex subunit 6 / APC6 / CDC16 homolog / CDC16Hs / Cyclosome subunit 6


Mass: 71747.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC16, ANAPC6 / Production host: unidentified baculovirus / References: UniProt: Q13042
#12: Protein Cell division cycle protein 27 homolog / Cell cycle / Anaphase-promoting complex subunit 3 / APC3 / CDC27 homolog / CDC27Hs / H-NUC


Mass: 91973.125 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC27, ANAPC3, D0S1430E, D17S978E / Production host: unidentified baculovirus / References: UniProt: P30260
#14: Protein Cell division cycle protein 23 homolog / Cell cycle / Anaphase-promoting complex subunit 8 / APC8 / Cyclosome subunit 8


Mass: 68921.031 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC23, ANAPC8 / Production host: unidentified baculovirus / References: UniProt: Q9UJX2
#15: Protein Cell division cycle protein 20 homolog / Cell cycle / p55CDC


Mass: 54796.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC20 / Production host: unidentified baculovirus / References: UniProt: Q12834

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box classCOMPLEX#1-#160MULTIPLE SOURCES
2Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box classCOMPLEX#1-#151RECOMBINANT
3Cryo-EM structure of the APC/C-Cdc20-Cdk2-cyclinA2-Cks2 complex, the D2 box classCOMPLEX#161RECOMBINANT
Molecular weightValue: 1.3 MDa
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22unidentified baculovirus10469
33Esacherichia coli562
Buffer solutionpH: 8 / Details: 20mM Hepes, 150mM NaCl, 0.5mM TCEP
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 28 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: RELION / Version: 3 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117044 / Symmetry type: POINT

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