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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 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 / mitotic nuclear membrane disassembly / Phosphorylation of Emi1 / Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes / cyclin A2-CDK1 complex / patched binding / meiotic spindle organization / Nuclear Pore Complex (NPC) Disassembly / Transcriptional regulation by RUNX2 / Phosphorylation of the APC/C / outer kinetochore / mitotic cell cycle phase transition / 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 / Golgi Cisternae Pericentriolar Stack Reorganization / cyclin-dependent protein serine/threonine kinase activator activity / Condensation of Prometaphase Chromosomes / response to copper ion / chromosome condensation / 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 G2 DNA damage checkpoint signaling / mitotic sister chromatid segregation / Regulation of APC/C activators between G1/S and early anaphase / regulation of embryonic development / cellular response to organic cyclic compound / mitotic cytokinesis / response to axon injury / cyclin-dependent protein kinase holoenzyme complex / chromosome organization / catalytic activity / cyclin-dependent kinase / animal organ regeneration / Nuclear events stimulated by ALK signaling in cancer / cyclin-dependent protein serine/threonine kinase activity / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / response to cadmium ion / Cyclin A/B1/B2 associated events during G2/M transition / cysteine-type peptidase activity / 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 / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / epithelial cell differentiation / Hsp70 protein binding / APC/C:Cdc20 mediated degradation of Cyclin B / Anchoring of the basal body to the plasma membrane / regulation of mitotic cell cycle / positive regulation of G2/M transition of mitotic cell cycle / ERK1 and ERK2 cascade / positive regulation of mitotic cell cycle / AURKA Activation by TPX2 / RNA polymerase II CTD heptapeptide repeat kinase activity / cyclin binding / Condensation of Prophase Chromosomes / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / mitotic spindle organization / APC/C:Cdc20 mediated degradation of Securin / positive regulation of DNA replication / response to activity / ubiquitin binding / molecular function activator activity / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / peptidyl-threonine phosphorylation 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 |