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Yorodumi- EMDB-12368: CryoEM structure of the human Separase-Cdk1-cyclin B1-Cks1 complex -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12368 | |||||||||
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Title | CryoEM structure of the human Separase-Cdk1-cyclin B1-Cks1 complex | |||||||||
Map data | Separase-Cdk1-cyclin B1-Cks1 complex (postprocessed). | |||||||||
Sample |
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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 / Golgi disassembly / microtubule cytoskeleton organization involved in mitosis / ventricular cardiac muscle cell development / G2/M DNA replication checkpoint / Depolymerization of the Nuclear Lamina / E2F-enabled inhibition of pre-replication complex formation / 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 / establishment of mitotic spindle localization / Activation of NIMA Kinases NEK9, NEK6, NEK7 / homologous chromosome segregation / mitotic nuclear membrane disassembly / Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes / Phosphorylation of Emi1 / cyclin A2-CDK1 complex / patched binding / meiotic spindle organization / Transcriptional regulation by RUNX2 / Nuclear Pore Complex (NPC) Disassembly / Phosphorylation of the APC/C / mitotic cell cycle phase transition / 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 / Initiation of Nuclear Envelope (NE) Reformation / protein localization to kinetochore / Polo-like kinase mediated events / cyclin-dependent protein serine/threonine kinase activator activity / Golgi Cisternae Pericentriolar Stack Reorganization / Condensation of Prometaphase Chromosomes / centrosome cycle / response to copper ion / [RNA-polymerase]-subunit kinase / chromosome condensation / cyclin-dependent protein serine/threonine kinase regulator activity / SCF ubiquitin ligase complex / mitotic metaphase chromosome alignment / cysteine-type endopeptidase inhibitor activity / cyclin-dependent protein kinase activity / G1/S-Specific Transcription / protein deubiquitination / MAPK3 (ERK1) activation / response to amine / ubiquitin-like protein ligase binding / mitotic G2 DNA damage checkpoint signaling / cellular response to organic cyclic compound / Regulation of APC/C activators between G1/S and early anaphase / mitotic sister chromatid segregation / mitotic cytokinesis / regulation of embryonic development / catalytic activity / response to axon injury / animal organ regeneration / chromosome organization / cyclin-dependent kinase / cyclin-dependent protein serine/threonine kinase activity / response to cadmium ion / Regulation of MITF-M-dependent genes involved in cell cycle and proliferation / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / Nuclear events stimulated by ALK signaling in cancer / Cyclin A/B1/B2 associated events during G2/M transition / cyclin-dependent protein kinase holoenzyme complex / positive regulation of cardiac muscle cell proliferation / cysteine-type peptidase activity / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / epithelial cell differentiation / regulation of mitotic cell cycle / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / positive regulation of G2/M transition of mitotic cell cycle / Resolution of Sister Chromatid Cohesion / Hsp70 protein binding / APC/C:Cdc20 mediated degradation of Cyclin B / ERK1 and ERK2 cascade / AURKA Activation by TPX2 / cyclin binding / positive regulation of mitotic cell cycle / RNA polymerase II CTD heptapeptide repeat kinase activity / mitotic spindle organization / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Condensation of Prophase Chromosomes / ubiquitin binding / positive regulation of DNA replication / response to activity Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
Authors | Yu J / Raia P / Ghent CM / Raisch T / Sadian Y / Barford D / Raunser S / Morgan DO / Boland A | |||||||||
Funding support | Switzerland, United States, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12368.map.gz | 12.1 MB | EMDB map data format | |
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Header (meta data) | emd-12368-v30.xml emd-12368.xml | 21.4 KB 21.4 KB | Display Display | EMDB header |
Images | emd_12368.png | 166.1 KB | ||
Others | emd_12368_additional_1.map.gz | 140.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12368 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12368 | HTTPS FTP |
-Validation report
Summary document | emd_12368_validation.pdf.gz | 212.3 KB | Display | EMDB validaton report |
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Full document | emd_12368_full_validation.pdf.gz | 211.4 KB | Display | |
Data in XML | emd_12368_validation.xml.gz | 6.9 KB | Display | |
Data in CIF | emd_12368_validation.cif.gz | 7.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12368 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12368 | HTTPS FTP |
-Related structure data
Related structure data | 7nj0MC 7nj1C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12368.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Separase-Cdk1-cyclin B1-Cks1 complex (postprocessed). | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.88 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: Separase-Cdk1-cyclin B1-Cks1 complex (unsharpened).
File | emd_12368_additional_1.map | ||||||||||||
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Annotation | Separase-Cdk1-cyclin B1-Cks1 complex (unsharpened). | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Mutual inhibitory complex of human separase-Cdk1-cyclin B1-Cks1 (...
Entire | Name: Mutual inhibitory complex of human separase-Cdk1-cyclin B1-Cks1 (CCC) complex. |
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Components |
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-Supramolecule #1: Mutual inhibitory complex of human separase-Cdk1-cyclin B1-Cks1 (...
Supramolecule | Name: Mutual inhibitory complex of human separase-Cdk1-cyclin B1-Cks1 (CCC) complex. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
-Macromolecule #1: Securin,Separin
Macromolecule | Name: Securin,Separin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: separase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 244.677328 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: MQLGPPSPVK MPSPPWESNL LQSPSSILST LDVELPPVCC DIDIGGSGGS GGGSGGGSGE NLYFQGGGSG GSGMRSFKRV NFGTLLSSQ KEAEELLPDL KEFLSNPPAG FPSSRSDAER RQACDAILRA CNQQLTAKLA CPRHLGSLLE LAELACDGYL V STPQRPPL ...String: MQLGPPSPVK MPSPPWESNL LQSPSSILST LDVELPPVCC DIDIGGSGGS GGGSGGGSGE NLYFQGGGSG GSGMRSFKRV NFGTLLSSQ KEAEELLPDL KEFLSNPPAG FPSSRSDAER RQACDAILRA CNQQLTAKLA CPRHLGSLLE LAELACDGYL V STPQRPPL YLERILFVLL RNAAAQGSPE VTLRLAQPLH ACLVQCSREA APQDYEAVAR GSFSLLWKGA EALLERRAAF AA RLKALSF LVLLEDESTP CEVPHFASPT ACRAVAAHQL FDASGHGLNE ADADFLDDLL SRHVIRALVG ERGSSSGLLS PQR ALCLLE LTLEHCRRFC WSRHHDKAIS AVEKAHSYLR NTNLAPSLQL CQLGVKLLQV GEEGPQAVAK LLIKASAVLS KSME APSPP LRALYESCQF FLSGLERGTK RRYRLDAILS LFAFLGGYCS LLQQLRDDGV YGGSSKQQQS FLQMYFQGLH LYTVV VYDF AQGCQIVDLA DLTQLVDSCK STVVWMLEAL EGLSGQELTD HMGMTASYTS NLAYSFYSHK LYAEACAISE PLCQHL GLV KPGTYPEVPP EKLHRCFRLQ VESLKKLGKQ AQGCKMVILW LAALQPCSPE HMAEPVTFWV RVKMDAARAG DKELQLK TL RDSLSGWDPE TLALLLREEL QAYKAVRADT GQERFNIICD LLELSPEETP AGAWARATHL VELAQVLCYH DFTQQTNC S ALDAIREALQ LLDSVRPEAQ ARDQLLDDKA QALLWLYICT LEAKIQEGIE RDRRAQAPGN LEEFEVNDLN YEDKLQEDR FLYSNIAFNL AADAAQSKCL DQALALWKEL LTKGQAPAVR CLQQTAASLQ ILAALYQLVA KPMQALEVLL LLRIVSERLK DHSKAAGSS CHITQLLLTL GCPSYAQLHL EEAASSLKHL DQTTDTYLLL SLTCDLLRSQ LYWTHQKVTK GVSLLLSVLR D PALQKSSK AWYLLRVQVL QLVAAYLSLP SNNLSHSLWE QLCAQGWQTP EIALIDSHKL LRSIILLLMG SDILSTQKAA VE TSFLDYG ENLVQKWQVL SEVLSCSEKL VCHLGRLGSV SEAKAFCLEA LKLTTKLQIP RQCALFLVLK GELELARNDI DLC QSDLQQ VLFLLESCTE FGGVTQHLDS VKKVHLQKGK QQAQVPCPPQ LPEEELFLRG PALELVATVA KEPGPIAPST NS (SEP)PVLKTK PQPIPNFLSH SPTCDCSLCA SPVLTAVCLR WVLVTAGVRL AMGHQAQGLD LLQVVLKGCP EAAERLTQA LQASLNHKTP PSLVPSLLDE ILAQAYTLLA LEGLNQPSNE SLQKVLQSGL KFVAARIPHL EPWRASLLLI WALTKLGGLS CCTTQLFAS SWGWQPPLIK SVPGSEPSKT QGQKRSGRGR QKLASAPLSL NNTSQKGLEG RGLPCTPKPP DRIRQAGPHV P FTVFEEVC PTESKPEVPQ APRVQQRVQT RLKVNFSDDS DLEDPVSAEA WLAEEPKRRG TASRGRGRAR KGLSLKTDAV VA PGSAPGN PGLNGRSRRA KKVASRHCEE RRPQRASDQA RPGPEIMRTI PEEELTDNWR KMSFEILRGS DGEDSASGGK TPA PGPEAA SGEWELLRLD SSKKKLPSPC PDKESDKDLG PRLQLPSAPV ATGLSTLDSI CDSLSVAFRG ISHCPPSGLY AHLC RFLAL CLGHRDPYAT AFLVTESVSI TCRHQLLTHL HRQLSKAQKH RGSLEIADQL QGLSLQEMPG DVPLARIQRL FSFRA LESG HFPQPEKESF QERLALIPSG VTVCVLALAT LQPGTVGNTL LLTRLEKDSP PVSVQIPTGQ NKLHLRSVLN EFDAIQ KAQ KENSSCTDKR EWWTGRLALD HRMEVLIASL EKSVLGCWKG LLLPSSEEPG PAQEASRLQE LLQDCGWKYP DRTLLKI ML SGAGALTPQD IQALAYGLCP TQPERAQELL NEAVGRLQGL TVPSNSHLVL VLDKDLQKLP WESMPSLQAL PVTRLPSF R FLLSYSIIKE YGASPVLSQG VDPRSTFYVL NPHNNLSSTE EQFRANFSSE AGWRGVVGEV PRPEQVQEAL TKHDLYIYA GHGAGARFLD GQAVLRLSCR AVALLFGSSS AALAVHGNLE GAGIVLKYIM AGCPLFLGNL WDVTDRDIDR YTEALLQGWL GAGPGAPLL YYVNQARQAP RLKYLIGAAP IAYGLPVSLR SSLAEENLYF QSWSHPQFEK GGGSGGGSGG GSWSHPQFEK |
-Macromolecule #2: Cyclin-dependent kinase 1
Macromolecule | Name: Cyclin-dependent kinase 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: cyclin-dependent kinase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 36.667098 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: MEDYTKIEKI GEGTYGVVYK GRHKTTGQVV AMKKIRLESE EEGVPSTAIR EISLLKELRH PNIVSLQDVL MQDSRLYLIF EFLSMDLKK YLDSIPPGQY MDSSLVKSYL YQILQGIVFC HSRRVLHRDL KPQNLLIDDK GTIKLADFGL ARAFGIPIRV Y (TPO)HEVVTLW ...String: MEDYTKIEKI GEGTYGVVYK GRHKTTGQVV AMKKIRLESE EEGVPSTAIR EISLLKELRH PNIVSLQDVL MQDSRLYLIF EFLSMDLKK YLDSIPPGQY MDSSLVKSYL YQILQGIVFC HSRRVLHRDL KPQNLLIDDK GTIKLADFGL ARAFGIPIRV Y (TPO)HEVVTLW YRSPEVLLGS ARYSTPVDIW SIGTIFAELA TKKPLFHGDS EIDQLFRIFR ALGTPNNEVW PEVESLQD Y KNTFPKWKPG SLASHVKNLD ENGLDLLSKM LIYDPAKRIS GKMALNHPYF NDLDNQIKKM IAAEALEVLF QGPHHHHHH HH |
-Macromolecule #3: G2/mitotic-specific cyclin-B1,G2/mitotic-specific cyclin-B1
Macromolecule | Name: G2/mitotic-specific cyclin-B1,G2/mitotic-specific cyclin-B1 type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 52.625723 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: MALRVTRNSK INAENKAKIN MAGAKRVPTA PAATSKPGLR PRTALGDIGN KVSEQLQAKM PMKKEAKPSA TGKVIDKKLP KPLEKVPML VPVPVSEPVP EPEPEPEPEP VKEEKLSPEP ILVDTASPSP METSGCAPAE EDLCQAFSDV ILAVNDVDAE D GADPNLCS ...String: MALRVTRNSK INAENKAKIN MAGAKRVPTA PAATSKPGLR PRTALGDIGN KVSEQLQAKM PMKKEAKPSA TGKVIDKKLP KPLEKVPML VPVPVSEPVP EPEPEPEPEP VKEEKLSPEP ILVDTASPSP METSGCAPAE EDLCQAFSDV ILAVNDVDAE D GADPNLCS EYVKDIYAYL RQLEEEQAVR PKYLLGREVT GNMRAILIDW LVQVQMKFRL LQETMYMTVS IIDRFMQNNC VP KKMLQLV GVTAMFIASK YEEMYPPEIG DFAFVTDNTY TKHQIRQMEM KILRALNFGL GRPLPLHFLR RASKIGEVDV EQH TLAKYL MELTMLDYDM VHFPPSQIAA GAFCLALKIL DNGEWTPTLQ HYLSYTEESL LPVMQHLAKN VVMVNQGLTK HMTV KNKYA TSKHAKISTL PQLNSALVQD LAKAVAKVSS LAEENLYFQS WSHPQFEKGG GSGGGSGGGS WSHPQFEK |
-Macromolecule #4: Cyclin-dependent kinases regulatory subunit 1
Macromolecule | Name: Cyclin-dependent kinases regulatory subunit 1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 9.679211 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: MSHKQIYYSD KYDDEEFEYR HVMLPKDIAK LVPKTHLMSE SEWRNLGVQQ SQGWVHYMIH EPEPHILLFR RPLPKKPKK |
-Macromolecule #5: PHOSPHATE ION
Macromolecule | Name: PHOSPHATE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: PO4 |
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Molecular weight | Theoretical: 94.971 Da |
Chemical component information | ChemComp-PO4: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.050 mg/mL |
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Buffer | pH: 7.8 |
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: LEICA EM GP |
Details | The sample was monodisperse. We use graphene oxide-coated EM grids. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 5 / Number real images: 13640 / Average exposure time: 3.0 sec. / Average electron dose: 78.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.3 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
CTF correction | Software: (Name: Gctf, cryoSPARC) |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 312836 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |