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- PDB-6tm5: Cryo-EM structure of the Anaphase-promoting complex/Cyclosome, in... -

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Entry
Database: PDB / ID: 6tm5
TitleCryo-EM structure of the Anaphase-promoting complex/Cyclosome, in complex with the Nek2A substrate at 3.9 angstrom resolution
Components
  • (Anaphase-promoting complex subunit ...) x 11
  • (Cell division cycle protein ...Cell cycle) x 3
  • (Serine/threonine-protein kinase ...Serine/threonine-specific protein kinase) x 2
  • Apc1 loop
KeywordsCELL CYCLE / E3 ubiquitin Ligase / Complex
Function / homology
Function and homology information


negative regulation of centriole-centriole cohesion / centrosome separation / regulation of mitotic cell cycle spindle assembly checkpoint / positive regulation of synapse maturation / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / regulation of attachment of spindle microtubules to kinetochore / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects ...negative regulation of centriole-centriole cohesion / centrosome separation / regulation of mitotic cell cycle spindle assembly checkpoint / positive regulation of synapse maturation / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / regulation of attachment of spindle microtubules to kinetochore / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / anaphase-promoting complex-dependent catabolic process / regulation of meiotic cell cycle / positive regulation of mitotic metaphase/anaphase transition / metaphase/anaphase transition of mitotic cell cycle / regulation of exit from mitosis / positive regulation of synaptic plasticity / Phosphorylation of the APC/C / protein K11-linked ubiquitination / regulation of mitotic centrosome separation / enzyme-substrate adaptor activity / regulation of mitotic metaphase/anaphase transition / positive regulation of dendrite morphogenesis / ubiquitin-ubiquitin ligase activity / regulation of mitotic nuclear division / mitotic metaphase chromosome alignment / cullin family protein binding / positive regulation of telomere capping / Regulation of APC/C activators between G1/S and early anaphase / blastocyst development / Transcriptional Regulation by VENTX / mitotic spindle assembly / spindle assembly / positive regulation of axon extension / heterochromatin / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / positive regulation of telomerase activity / Recruitment of NuMA to mitotic centrosomes / positive regulation of telomere maintenance via telomerase / APC/C:Cdc20 mediated degradation of Cyclin B / Anchoring of the basal body to the plasma membrane / regulation of mitotic cell cycle / APC-Cdc20 mediated degradation of Nek2A / nuclear periphery / AURKA Activation by TPX2 / meiotic cell cycle / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / condensed nuclear chromosome / chromosome segregation / Assembly of the pre-replicative complex / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / brain development / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / mitotic spindle / spindle / kinetochore / CDK-mediated phosphorylation and removal of Cdc6 / spindle pole / ubiquitin-protein transferase activity / Separation of Sister Chromatids / ubiquitin protein ligase activity / microtubule cytoskeleton / Regulation of PLK1 Activity at G2/M Transition / Antigen processing: Ubiquitination & Proteasome degradation / mitotic cell cycle / nervous system development / midbody / ubiquitin-dependent protein catabolic process / Senescence-Associated Secretory Phenotype (SASP) / protein phosphatase binding / microtubule / protein autophosphorylation / cell differentiation / molecular adaptor activity / protein ubiquitination / non-specific serine/threonine protein kinase / protein kinase activity / cell cycle / cell division / negative regulation of gene expression / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / ubiquitin protein ligase binding / nucleolus / protein-containing complex / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Anaphase-promoting complex subunit 15 / Anaphase-promoting complex subunit 15 / Anaphase-promoting complex subunit 4, metazoa / : / Anaphase-promoting complex subunit 5, N-terminal domain / Anaphase-promoting complex subunit 1, C-terminal / Anaphase-promoting complex subunit 1, middle domain / : / : ...: / Anaphase-promoting complex subunit 15 / Anaphase-promoting complex subunit 15 / Anaphase-promoting complex subunit 4, metazoa / : / Anaphase-promoting complex subunit 5, N-terminal domain / Anaphase-promoting complex subunit 1, C-terminal / Anaphase-promoting complex subunit 1, middle domain / : / : / Anaphase-promoting complex subunit 1 WD40 beta-propeller domain / Anaphase-promoting complex sub unit 1 C-terminal domain / Anaphase-promoting complex subunit 1 middle domain / APC1 beta sandwich domain / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex, subunit 16 / Cdc23 / Apc13 / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 4 long domain / Anaphase promoting complex subunit 8 / Cdc23 / Apc13p protein / Anaphase-promoting complex, cyclosome, subunit 4 / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 5 domain / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit APC10/Doc1 / Anaphase-promoting complex, subunit CDC26 / Anaphase-promoting complex APC subunit CDC26 / Anaphase-promoting complex subunit 11, RING-H2 finger / Anaphase-promoting complex subunit 11 RING-H2 finger / Anaphase-promoting complex subunit 2, C-terminal / Anaphase-promoting complex subunit 2 / Anaphase promoting complex (APC) subunit 2 / Anaphase promoting complex (APC) subunit 2 / Anaphase-promoting complex, cyclosome, subunit 3 / APC10/DOC domain / Anaphase-promoting complex, subunit 10 (APC10) / DOC domain profile. / Anaphase-promoting complex, subunit 10 (APC10) / TPR repeat / Tetratricopeptide repeat / Anaphase-promoting complex subunit 4, WD40 domain / Anaphase-promoting complex subunit 4 WD40 domain / Tetratricopeptide repeat / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Tetratricopeptide repeat 1 / Tetratricopeptide repeat / Tetratricopeptide repeat domain / Tetratricopeptide repeat / Zinc/RING finger domain, C3HC4 (zinc finger) / Herpes Virus-1 / Galactose-binding domain-like / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Galactose-binding-like domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Armadillo-like helical / Alpha Horseshoe / Tetratricopeptide-like helical domain superfamily / Zinc finger, RING/FYVE/PHD-type / Winged helix DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / Jelly Rolls / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40/YVTN repeat-like-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Sandwich / 2-Layer Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Cell division cycle protein 27 homolog / Serine/threonine-protein kinase Nek2 / Anaphase-promoting complex subunit 15 / Cell division cycle protein 16 homolog / Anaphase-promoting complex subunit CDC26 / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 11 / Cell division cycle protein 23 homolog ...Cell division cycle protein 27 homolog / Serine/threonine-protein kinase Nek2 / Anaphase-promoting complex subunit 15 / Cell division cycle protein 16 homolog / Anaphase-promoting complex subunit CDC26 / Anaphase-promoting complex subunit 16 / Anaphase-promoting complex subunit 13 / Anaphase-promoting complex subunit 1 / Anaphase-promoting complex subunit 11 / Cell division cycle protein 23 homolog / Anaphase-promoting complex subunit 7 / Anaphase-promoting complex subunit 5 / Anaphase-promoting complex subunit 4 / Anaphase-promoting complex subunit 2 / Anaphase-promoting complex subunit 10
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsAlfieri, C. / Barford, D.
Funding support1items
OrganizationGrant numberCountry
Cancer Research UK
CitationJournal: EMBO Rep / Year: 2020
Title: A unique binding mode of Nek2A to the APC/C allows its ubiquitination during prometaphase.
Authors: Claudio Alfieri / Thomas Tischer / David Barford /
Abstract: The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of ...The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle-specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational-specific binder of the APC/C-MCC complex (APC/C ) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase-specific ubiquitination.
History
DepositionDec 3, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 17, 2020Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

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Assembly

Deposited unit
A: Anaphase-promoting complex subunit 1
B: Anaphase-promoting complex subunit 11
C: Cell division cycle protein 23 homolog
D: Anaphase-promoting complex subunit 15
E: Anaphase-promoting complex subunit 16
F: Cell division cycle protein 27 homolog
G: Anaphase-promoting complex subunit CDC26
H: Cell division cycle protein 27 homolog
I: Anaphase-promoting complex subunit 4
J: Cell division cycle protein 16 homolog
K: Cell division cycle protein 16 homolog
L: Anaphase-promoting complex subunit 10
M: Anaphase-promoting complex subunit 13
N: Anaphase-promoting complex subunit 2
O: Anaphase-promoting complex subunit 5
P: Cell division cycle protein 23 homolog
T: Apc1 loop
W: Anaphase-promoting complex subunit CDC26
X: Anaphase-promoting complex subunit 7
Y: Anaphase-promoting complex subunit 7
S: Serine/threonine-protein kinase Nek2
Q: Serine/threonine-protein kinase Nek2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,277,49025
Polymers1,277,29422
Non-polymers1963
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Anaphase-promoting complex subunit ... , 11 types, 13 molecules ABDEGWILMNOXY

#1: Protein Anaphase-promoting complex subunit 1 / / APC1 / Cyclosome subunit 1 / Mitotic checkpoint regulator / Testis-specific gene 24 protein


Mass: 216775.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC1, TSG24 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9H1A4
#2: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NYG5
#4: 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: Trichoplusia ni (cabbage looper) / References: UniProt: P60006
#5: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q96DE5
#7: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q8NHZ8
#8: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UJX5
#10: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UM13
#11: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BS18
#12: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UJX6
#13: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UJX4
#15: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UJX3

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Cell division cycle protein ... , 3 types, 6 molecules CPFHJK

#3: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UJX2
#6: 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: Trichoplusia ni (cabbage looper) / References: UniProt: P30260
#9: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q13042

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Serine/threonine-protein kinase ... , 2 types, 2 molecules SQ

#16: Protein Serine/threonine-protein kinase Nek2 / HSPK 21 / Never in mitosis A-related kinase 2 / NimA-related protein kinase 2 / NimA-like protein kinase 1


Mass: 51788.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NEK2, NEK2A, NLK1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P51955, non-specific serine/threonine protein kinase
#17: Protein Serine/threonine-protein kinase Nek2 / HSPK 21 / Never in mitosis A-related kinase 2 / NimA-related protein kinase 2 / NimA-like protein kinase 1


Mass: 51848.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NEK2, NEK2A, NLK1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P51955, non-specific serine/threonine protein kinase

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Protein/peptide / Non-polymers , 2 types, 4 molecules T

#14: Protein/peptide Apc1 loop


Mass: 1183.313 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)
#18: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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

ComponentName: Cryo-EM structure of the Anaphase-promoting complex/Cyclosome, in complex with the Nek2A substrate at 3.9 angstrom resolution
Type: COMPLEX / Entity ID: #1-#17 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding 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: 29 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233840 / Symmetry type: POINT
RefinementHighest resolution: 3.9 Å

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