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- PDB-7nj0: CryoEM structure of the human Separase-Cdk1-cyclin B1-Cks1 complex -

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Basic information

Entry
Database: PDB / ID: 7nj0
TitleCryoEM structure of the human Separase-Cdk1-cyclin B1-Cks1 complex
Components
  • Cyclin-dependent kinase 1
  • Cyclin-dependent kinases regulatory subunit 1
  • G2/mitotic-specific cyclin-B1,G2/mitotic-specific cyclin-B1
  • Securin,Separin
KeywordsHYDROLASE / autoinhibition phosphate-binding pocket pseudosubstrate Scc1
Function / homology
Function and homology information


negative regulation of mitotic sister chromatid separation / negative regulation of sister chromatid cohesion / separase / regulation of Schwann cell differentiation / pronuclear fusion / cyclin B1-CDK1 complex / positive regulation of mitochondrial ATP synthesis coupled electron transport / Mitotic Prophase / positive regulation of mitotic sister chromatid segregation / meiotic chromosome separation ...negative regulation of mitotic sister chromatid separation / negative regulation of sister chromatid cohesion / separase / regulation of Schwann cell differentiation / pronuclear fusion / cyclin B1-CDK1 complex / positive regulation of mitochondrial ATP synthesis coupled electron transport / Mitotic Prophase / positive regulation of mitotic sister chromatid segregation / meiotic chromosome separation / histone kinase activity / Golgi disassembly / microtubule cytoskeleton organization involved in mitosis / G2/M DNA replication checkpoint / E2F-enabled inhibition of pre-replication complex formation / ventricular cardiac muscle cell development / Depolymerization of the Nuclear Lamina / positive regulation of attachment of spindle microtubules to kinetochore / MASTL Facilitates Mitotic Progression / regulation of mitotic cell cycle spindle assembly checkpoint / establishment of mitotic spindle localization / Activation of NIMA Kinases NEK9, NEK6, NEK7 / homologous chromosome segregation / Phosphorylation of Emi1 / Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes / patched binding / cyclin A2-CDK1 complex / meiotic spindle organization / positive regulation of mitotic metaphase/anaphase transition / Nuclear Pore Complex (NPC) Disassembly / Transcriptional regulation by RUNX2 / outer kinetochore / Phosphorylation of the APC/C / mitotic cell cycle phase transition / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / Initiation of Nuclear Envelope (NE) Reformation / protein localization to kinetochore / Polo-like kinase mediated events / Golgi Cisternae Pericentriolar Stack Reorganization / cyclin-dependent protein serine/threonine kinase activator activity / chromosome condensation / Condensation of Prometaphase Chromosomes / response to copper ion / centrosome cycle / [RNA-polymerase]-subunit kinase / cyclin-dependent protein serine/threonine kinase regulator activity / SCF ubiquitin ligase complex / mitotic metaphase chromosome alignment / cysteine-type endopeptidase inhibitor activity / G1/S-Specific Transcription / cyclin-dependent protein kinase activity / MAPK3 (ERK1) activation / response to amine / ubiquitin-like protein ligase binding / mitotic sister chromatid segregation / mitotic G2 DNA damage checkpoint signaling / regulation of embryonic development / Regulation of APC/C activators between G1/S and early anaphase / mitotic cytokinesis / cellular response to organic cyclic compound / chromosome organization / cyclin-dependent protein kinase holoenzyme complex / response to axon injury / cyclin-dependent kinase / animal organ regeneration / cyclin-dependent protein serine/threonine kinase activity / response to cadmium ion / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / cysteine-type peptidase activity / catalytic activity / Cyclin A/B1/B2 associated events during G2/M transition / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / positive regulation of cardiac muscle cell proliferation / Recruitment of mitotic centrosome proteins and complexes / ERK1 and ERK2 cascade / Hsp70 protein binding / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / epithelial cell differentiation / APC/C:Cdc20 mediated degradation of Cyclin B / Anchoring of the basal body to the plasma membrane / positive regulation of G2/M transition of mitotic cell cycle / regulation of mitotic cell cycle / cyclin binding / positive regulation of mitotic cell cycle / RNA polymerase II CTD heptapeptide repeat kinase activity / AURKA Activation by TPX2 / mitotic spindle organization / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Condensation of Prophase Chromosomes / positive regulation of DNA replication / response to activity / ubiquitin binding / APC/C:Cdc20 mediated degradation of Securin / molecular function activator activity / spindle microtubule / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / peptidyl-threonine phosphorylation / G1/S transition of mitotic cell cycle
Similarity search - Function
Securin sister-chromatid separation inhibitor / Securin sister-chromatid separation inhibitor / Peptidase C50, separase / SEPARIN core domain / SEPARIN core domain profile. / : / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. ...Securin sister-chromatid separation inhibitor / Securin sister-chromatid separation inhibitor / Peptidase C50, separase / SEPARIN core domain / SEPARIN core domain profile. / : / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. / Cyclin-dependent kinases regulatory subunits signature 2. / Cyclin-dependent kinase regulatory subunit / : / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / Cyclin, C-terminal domain / Cyclin_C / Cyclin, N-terminal / Cyclin, N-terminal domain / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
PHOSPHATE ION / Securin / Cyclin-dependent kinase 1 / G2/mitotic-specific cyclin-B1 / Cyclin-dependent kinases regulatory subunit 1 / Separin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsYu, J. / Raia, P. / Ghent, C.M. / Raisch, T. / Sadian, Y. / Barford, D. / Raunser, S. / Morgan, D.O. / Boland, A.
Funding support Switzerland, United States, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_185235 Switzerland
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM118053 United States
CitationJournal: Nature / Year: 2021
Title: Structural basis of human separase regulation by securin and CDK1-cyclin B1.
Authors: Jun Yu / Pierre Raia / Chloe M Ghent / Tobias Raisch / Yashar Sadian / Simone Cavadini / Pramod M Sabale / David Barford / Stefan Raunser / David O Morgan / Andreas Boland /
Abstract: In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase ...In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD21). Separase is activated by degradation of its inhibitors, securin and cyclin B, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans and yeast, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK1. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.
History
DepositionFeb 14, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 4, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 18, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Securin,Separin
B: Cyclin-dependent kinase 1
C: G2/mitotic-specific cyclin-B1,G2/mitotic-specific cyclin-B1
D: Cyclin-dependent kinases regulatory subunit 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)343,7445
Polymers343,6494
Non-polymers951
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12410 Å2
ΔGint-66 kcal/mol
Surface area73870 Å2
MethodPISA

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Components

#1: Protein Securin,Separin / Esp1-associated protein / Pituitary tumor-transforming gene 1 protein / Tumor-transforming protein ...Esp1-associated protein / Pituitary tumor-transforming gene 1 protein / Tumor-transforming protein 1 / hPTTG / Caspase-like protein ESPL1 / Extra spindle poles-like 1 protein / Separase


Mass: 244677.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PTTG1, EAP1, PTTG, TUTR1, ESPL1, ESP1, KIAA0165 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O95997, UniProt: Q14674, separase
#2: Protein Cyclin-dependent kinase 1 / / CDK1 / Cell division control protein 2 homolog / Cell division protein kinase 1 / p34 protein kinase


Mass: 36667.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDK1, CDC2, CDC28A, CDKN1, P34CDC2 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P06493, cyclin-dependent kinase, [RNA-polymerase]-subunit kinase
#3: Protein G2/mitotic-specific cyclin-B1,G2/mitotic-specific cyclin-B1


Mass: 52625.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCNB1, CCNB / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P14635
#4: Protein Cyclin-dependent kinases regulatory subunit 1 / CKS-1


Mass: 9679.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CKS1B, CKS1, PNAS-143, PNAS-16 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P61024
#5: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
Has ligand of interestY

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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: Mutual inhibitory complex of human separase-Cdk1-cyclin B1-Cks1 (CCC) complex.
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.8
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was monodisperse. We use graphene oxide-coated EM grids.
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K

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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 / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Calibrated defocus min: 1300 nm / Calibrated defocus max: 2500 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 78 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 5 / Num. of real images: 13640

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Processing

SoftwareName: PHENIX / Version: 1.18rc5_3822: / Classification: refinement
EM software
IDNameCategory
2Topazparticle selection
3EPUimage acquisition
5GctfCTF correction
6cryoSPARCCTF correction
9Cootmodel fitting
10UCSF Chimeramodel fitting
15RELION3D reconstruction
16PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 312836 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model building
IDPDB-ID 3D fitting-ID
11QMZ

1qmz

1
24YC3

4yc3

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414638
ELECTRON MICROSCOPYf_angle_d0.59519883
ELECTRON MICROSCOPYf_dihedral_angle_d14.6221964
ELECTRON MICROSCOPYf_chiral_restr0.0392285
ELECTRON MICROSCOPYf_plane_restr0.0052528

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