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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 sister chromatid cohesion / separase / regulation of Schwann cell differentiation / cyclin A1-CDK1 complex / regulation of attachment of mitotic spindle microtubules to kinetochore / pronuclear fusion / cyclin B1-CDK1 complex / positive regulation of mitochondrial ATP synthesis coupled electron transport / Mitotic Prophase / positive regulation of mitotic sister chromatid segregation ...negative regulation of sister chromatid cohesion / separase / regulation of Schwann cell differentiation / cyclin A1-CDK1 complex / regulation of attachment of mitotic spindle microtubules to kinetochore / 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 / mitotic cell cycle phase transition / Golgi disassembly / microtubule cytoskeleton organization involved in mitosis / ventricular cardiac muscle cell development / G2/M DNA replication checkpoint / E2F-enabled inhibition of pre-replication complex formation / Depolymerization of the Nuclear Lamina / positive regulation of attachment of spindle microtubules to kinetochore / MASTL Facilitates Mitotic Progression / mitotic sister chromatid separation / regulation of mitotic cell cycle spindle assembly checkpoint / Activation of NIMA Kinases NEK9, NEK6, NEK7 / homologous chromosome segregation / mitotic nuclear membrane disassembly / : / cyclin A2-CDK1 complex / Phosphorylation of Emi1 / patched binding / establishment of mitotic spindle localization / meiotic spindle organization / Transcriptional regulation by RUNX2 / Nuclear Pore Complex (NPC) Disassembly / Phosphorylation of the APC/C / outer kinetochore / positive regulation of mitotic metaphase/anaphase transition / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / protein localization to kinetochore / RNA polymerase II CTD heptapeptide repeat Y1 kinase activity / RNA polymerase II CTD heptapeptide repeat S2 kinase activity / RNA polymerase II CTD heptapeptide repeat T4 kinase activity / RNA polymerase II CTD heptapeptide repeat S5 kinase activity / RNA polymerase II CTD heptapeptide repeat S7 kinase activity / Initiation of Nuclear Envelope (NE) Reformation / Polo-like kinase mediated events / response to copper ion / cyclin-dependent protein serine/threonine kinase activator activity / Golgi Cisternae Pericentriolar Stack Reorganization / Condensation of Prometaphase Chromosomes / centrosome cycle / chromosome condensation / [RNA-polymerase]-subunit kinase / cyclin-dependent protein serine/threonine kinase regulator activity / SCF ubiquitin ligase complex / mitotic metaphase chromosome alignment / MAPK3 (ERK1) activation / cyclin-dependent protein kinase activity / response to amine / G1/S-Specific Transcription / mitotic G2 DNA damage checkpoint signaling / microtubule organizing center / : / Regulation of APC/C activators between G1/S and early anaphase / ubiquitin-like protein ligase binding / mitotic cytokinesis / mitotic sister chromatid segregation / catalytic activity / regulation of embryonic development / cysteine-type endopeptidase inhibitor activity / protein deubiquitination / response to axon injury / chromosome organization / cyclin-dependent kinase / cyclin-dependent protein serine/threonine kinase activity / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / Regulation of MITF-M-dependent genes involved in cell cycle and proliferation / Cyclin A/B1/B2 associated events during G2/M transition / response to cadmium ion / Nuclear events stimulated by ALK signaling in cancer / cyclin-dependent protein kinase holoenzyme complex / positive regulation of cardiac muscle cell proliferation / positive regulation of G2/M transition of mitotic cell cycle / epithelial cell differentiation / RNA polymerase II CTD heptapeptide repeat kinase activity / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / peptidyl-threonine phosphorylation / ERK1 and ERK2 cascade / Recruitment of mitotic centrosome proteins and complexes / Hsp70 protein binding / APC/C:Cdc20 mediated degradation of Cyclin B / positive regulation of mitotic cell cycle / cysteine-type peptidase activity / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / Resolution of Sister Chromatid Cohesion / cyclin binding / regulation of mitotic cell cycle / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest
Similarity search - Function
Securin sister-chromatid separation inhibitor / Securin sister-chromatid separation inhibitor / Peptidase C50, separase / SEPARIN core domain / Peptidase family C50 / SEPARIN core domain profile. / : / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit ...Securin sister-chromatid separation inhibitor / Securin sister-chromatid separation inhibitor / Peptidase C50, separase / SEPARIN core domain / Peptidase family C50 / 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 / : / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / 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 / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
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
Revision 1.2Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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
Water00
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


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

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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 FIELD / 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-IDAccession codeInitial refinement model-IDSource nameType
11QMZ

1qmz

11QMZ1PDBexperimental model
24YC3

4yc3

14YC32PDBexperimental model
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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