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- EMDB-52303: Cryo-EM structure of human separase bound to SCC1 (310-550 aa) -

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Basic information

Entry
Database: EMDB / ID: EMD-52303
TitleCryo-EM structure of human separase bound to SCC1 (310-550 aa)
Map dataComposite map (postprocessed) of human separase bound to SCC1 (310-550 aa)
Sample
  • Complex: Separase bound to SCC1 (310-550 aa)
    • Protein or peptide: Double-strand-break repair protein rad21 homolog
    • Protein or peptide: Separin
  • Ligand: ZINC ION
KeywordsSeparase / cell cycle / SCC1 / RAD21 / protease / chromosome segregation / Auto-cleavage / SA1/2 / cohesin
Function / homology
Function and homology information


negative regulation of sister chromatid cohesion / separase / meiotic chromosome separation / negative regulation of mitotic metaphase/anaphase transition / Cohesin Loading onto Chromatin / meiotic cohesin complex / Establishment of Sister Chromatid Cohesion / establishment of meiotic sister chromatid cohesion / cohesin complex / mitotic cohesin complex ...negative regulation of sister chromatid cohesion / separase / meiotic chromosome separation / negative regulation of mitotic metaphase/anaphase transition / Cohesin Loading onto Chromatin / meiotic cohesin complex / Establishment of Sister Chromatid Cohesion / establishment of meiotic sister chromatid cohesion / cohesin complex / mitotic cohesin complex / positive regulation of sister chromatid cohesion / mitotic sister chromatid separation / negative regulation of glial cell apoptotic process / homologous chromosome segregation / establishment of mitotic spindle localization / negative regulation of G2/M transition of mitotic cell cycle / meiotic spindle organization / establishment of mitotic sister chromatid cohesion / replication-born double-strand break repair via sister chromatid exchange / chromatin looping / positive regulation of mitotic metaphase/anaphase transition / reciprocal meiotic recombination / negative regulation of interleukin-1 beta production / sister chromatid cohesion / lncRNA binding / mitotic cytokinesis / catalytic activity / mitotic sister chromatid segregation / positive regulation of interleukin-10 production / negative regulation of tumor necrosis factor production / chromosome, centromeric region / SUMOylation of DNA damage response and repair proteins / cis-regulatory region sequence-specific DNA binding / protein localization to chromatin / cysteine-type peptidase activity / Meiotic synapsis / Resolution of Sister Chromatid Cohesion / condensed nuclear chromosome / chromosome segregation / nuclear matrix / spindle pole / mitotic spindle / Separation of Sister Chromatids / double-strand break repair / chromosome / DNA recombination / midbody / Estrogen-dependent gene expression / DNA-binding transcription factor binding / negative regulation of neuron apoptotic process / response to hypoxia / cell division / cysteine-type endopeptidase activity / apoptotic process / chromatin binding / regulation of transcription by RNA polymerase II / centrosome / chromatin / proteolysis / nucleoplasm / membrane / nucleus / cytoplasm / cytosol
Similarity search - Function
Peptidase C50, separase / SEPARIN core domain / Separin, protease domain / SEPARIN core domain profile. / : / Rad21/Rec8-like protein, C-terminal, eukaryotic / Rad21/Rec8-like protein, N-terminal / Rad21/Rec8-like protein / Conserved region of Rad21 / Rec8 like protein / N terminus of Rad21 / Rec8 like protein ...Peptidase C50, separase / SEPARIN core domain / Separin, protease domain / SEPARIN core domain profile. / : / Rad21/Rec8-like protein, C-terminal, eukaryotic / Rad21/Rec8-like protein, N-terminal / Rad21/Rec8-like protein / Conserved region of Rad21 / Rec8 like protein / N terminus of Rad21 / Rec8 like protein / ScpA-like, C-terminal / Winged helix DNA-binding domain superfamily
Similarity search - Domain/homology
Double-strand-break repair protein rad21 homolog / Separin
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYu J / Schmidt S / Botto M / Boland A
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_185235 Switzerland
Swiss National Science FoundationTMSGI3_211581 Switzerland
CitationJournal: Sci Adv / Year: 2025
Title: Substrate recognition by human separase.
Authors: Jun Yu / Sophia Schmidt / Margherita Botto / Kitaik Lee / Chloe M Ghent / Jonah M Goodfried / Andrew Howe / Francis J O'Reilly / David O Morgan / Andreas Boland /
Abstract: The cohesin complex encircles sister chromatids in early mitosis. At anaphase onset, sister separation is triggered by the proteolytic cleavage of the cohesin subunit SCC1/RAD21 by separase. SCC1 ...The cohesin complex encircles sister chromatids in early mitosis. At anaphase onset, sister separation is triggered by the proteolytic cleavage of the cohesin subunit SCC1/RAD21 by separase. SCC1 contains two cleavage sites, where cleavage is stimulated by SCC1 phosphorylation. Substrate recognition and cleavage are only partly understood. Here, we determined structures of human separase in apo- or substrate-bound forms that, together with biochemical analysis, provide critical insights into separase cleavage regulation. We verify the first SCC1 cleavage site and reassign the second. We show that substrates, including separase autocleavage sites and the two SCC1 cleavage sites, interact with docking sites in separase, including five phosphate-binding sites. We also describe the interaction between the cohesin subunit SA1/SA2 and separase, which promotes cleavage at the second SCC1 site. Using cross-linking mass spectrometry and cryo-electron microscopy, we propose how cohesin is targeted by human separase. Our work provides an extensive functional and structural framework that explains a key event in cell division.
History
DepositionDec 10, 2024-
Header (metadata) releaseSep 3, 2025-
Map releaseSep 3, 2025-
UpdateMay 6, 2026-
Current statusMay 6, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52303.png

Downloads & links

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Map

FileDownload / File: emd_52303.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map (postprocessed) of human separase bound to SCC1 (310-550 aa)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 400 pix.
= 360.96 Å		0.9 Å/pix.
x 400 pix.
= 360.96 Å		0.9 Å/pix.
x 400 pix.
= 360.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9024 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.2
Minimum - Maximum-0.17138055 - 22.506640000000001
Average (Standard dev.)-0.018303907 (±0.37481028)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 360.96002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Composite map (unsharpened) of human separase bound to...

Fileemd_52303_additional_1.map
AnnotationComposite map (unsharpened) of human separase bound to SCC1 (310-550 aa)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Separase bound to SCC1 (310-550 aa)

EntireName: Separase bound to SCC1 (310-550 aa)
Components
  • Complex: Separase bound to SCC1 (310-550 aa)
    • Protein or peptide: Double-strand-break repair protein rad21 homolog
    • Protein or peptide: Separin
  • Ligand: ZINC ION

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Supramolecule #1: Separase bound to SCC1 (310-550 aa)

SupramoleculeName: Separase bound to SCC1 (310-550 aa) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: Double-strand-break repair protein rad21 homolog

MacromoleculeName: Double-strand-break repair protein rad21 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.693211 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: DITVKETKAK RKRKLIVDSV KELDSKTIRA QLSDYSDIVT TLDLAPPTKK LMMWKETGGV EKLFSLPAQP LWNNRLLKLF TRCLTPLVP EDLRKRRKGG EADNLDEFLK EFENPEVPRE DQQQQHQQRD VIDEPIIEEP SRLQESVMEA SRTNIDESAM P PPPPQGVK ...String:
DITVKETKAK RKRKLIVDSV KELDSKTIRA QLSDYSDIVT TLDLAPPTKK LMMWKETGGV EKLFSLPAQP LWNNRLLKLF TRCLTPLVP EDLRKRRKGG EADNLDEFLK EFENPEVPRE DQQQQHQQRD VIDEPIIEEP SRLQESVMEA SRTNIDESAM P PPPPQGVK RKAGQIDPEP VMPPQQVEQM EIPPVELPPE EPPNICQLIP ELELLPEKEK EKEKEKEDDE EEEDEDASGG DQ D

UniProtKB: Double-strand-break repair protein rad21 homolog

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Macromolecule #2: Separin

MacromoleculeName: Separin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: separase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 238.086344 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDYKDHDGDY KDHDIDYKDD DDKSGPGGSG GSGGGSGGGS GENLYFQGGG SGGSGMRSFK RVNFGTLLSS QKEAEELLPD LKEFLSNPP AGFPSSRSDA ERRQACDAIL RACNQQLTAK LACPRHLGSL LELAELACDG YLVSTPQRPP LYLERILFVL L RNAAAQGS ...String:
MDYKDHDGDY KDHDIDYKDD DDKSGPGGSG GSGGGSGGGS GENLYFQGGG SGGSGMRSFK RVNFGTLLSS QKEAEELLPD LKEFLSNPP AGFPSSRSDA ERRQACDAIL RACNQQLTAK LACPRHLGSL LELAELACDG YLVSTPQRPP LYLERILFVL L RNAAAQGS PEVTLRLAQP LHACLVQCSR EAAPQDYEAV ARGSFSLLWK GAEALLERRA AFAARLKALS FLVLLEDEST PC EVPHFAS PTACRAVAAH QLFDASGHGL NEADADFLDD LLSRHVIRAL VGERGSSSGL LSPQRALCLL ELTLEHCRRF CWS RHHDKA ISAVEKAHSY LRNTNLAPSL QLCQLGVKLL QVGEEGPQAV AKLLIKASAV LSKSMEAPSP PLRALYESCQ FFLS GLERG TKRRYRLDAI LSLFAFLGGY CSLLQQLRDD GVYGGSSKQQ QSFLQMYFQG LHLYTVVVYD FAQGCQIVDL ADLTQ LVDS CKSTVVWMLE ALEGLSGQEL TDHMGMTASY TSNLAYSFYS HKLYAEACAI SEPLCQHLGL VKPGTYPEVP PEKLHR CFR LQVESLKKLG KQAQGCKMVI LWLAALQPCS PEHMAEPVTF WVRVKMDAAR AGDKELQLKT LRDSLSGWDP ETLALLL RE ELQAYKAVRA DTGQERFNII CDLLELSPEE TPAGAWARAT HLVELAQVLC YHDFTQQTNC SALDAIREAL QLLDSVRP E AQARDQLLDD KAQALLWLYI CTLEAKMQEG IERDRRAQAP GNLEEFEVND LNYEDKLQED RFLYSNIAFN LAADAAQSK CLDQALALWK ELLTKGQAPA VRCLQQTAAS LQILAALYQL VAKPMQALEV LLLLRIVSER LKDHSKAAGS SCHITQLLLT LGCPSYAQL HLEEAASSLK HLDQTTDTYL LLSLTCDLLR SQLYWTHQKV TKGVSLLLSV LRDPALQKSS KAWYLLRVQV L QLVAAYLS LPSNNLSHSL WEQLCAQGWQ TPEIALIDSH KLLRSIILLL MGSDILSTQK AAVETSFLDY GENLVQKWQV LS EVLSCSE KLVCHLGRLG SVSEAKAFCL EALKLTTKLQ IPRQCALFLV LKGELELARN DIDLCQSDLQ QVLFLLESCT EFG GVTQHL DSVKKVHLQK GKQQAQVPCP PQLPEEELFL RGPALELVAT VAKEPGPIAP STNSSPVLKT KPQPIPNFLS HSPT CDCSL CASPVLTAVC LRWVLVTAGV RLAMGHQAQG LDLLQVVLKG CPEAAERLTQ ALQASLNHKT PPSLVPSLLD EILAQ AYTL LALEGLNQPS NESLQKVLQS GLKFVAARIP HLEPWRASLL LIWALTKLGG LSCCTTQLFA SSWGWQPPLI KSVPGS EPS KTQGQKRSGR GRQKLASAPL SLNNTSQKGL EGRGLPCTPK PPDRIRQAGP HVPFTVFEEV CPTESKPEVP QAPRVQQ RV QTRLKVNFSD DSDLEDPVSA EAWLAEEPKR RGTASRGRGR ARKGLSLKTD AVVAPGSAPG NPGLNGRSRR AKKVASRH C EERRPQRASD QARPGGLEVL FQGPGSGDSS KKKLPSPCPD KESDKDLGPR LQLPSAPVAT GLSTLDSICD SLSVAFRGI SHCPPSGLYA HLCRFLALCL GHRDPYATAF LVTESVSITC RHQLLTHLHR QLSKAQKHRG SLEIADQLQG LSLQEMPGDV PLARIQRLF SFRALESGHF PQPEKESFQE RLALIPSGVT VCVLALATLQ PGTVGNTLLL TRLEKDSPPV SVQIPTGQNK L HLRSVLNE FDAIQKAQKE NSSCTDKREW WTGRLALDHR MEVLIASLEK SVLGCWKGLL LPSSEEPGPA QEASRLQELL QD CGWKYPD RTLLKIMLSG AGALTPQDIQ ALAYGLCPTQ PERAQELLNE AVGRLQGLTV PSNSHLVLVL DKDLQKLPWE SMP SLQALP VTRLPSFRFL LSYSIIKEYG ASPVLSQGVD PRSTFYVLNP HNNLSSTEEQ FRANFSSEAG WRGVVGEVPR PEQV QEALT KHDLYIYAGH GAGARFLDGQ AVLRLSCRAV ALLFGSSSAA LAVHGNLEGA GIVLKYIMAG CPLFLGNLWD VTDRD IDRY TEALLQGWLG AGPGAPLLYY VNQARQAPRL KYLIGAAPIA YGLPVSLRSS LAEENLYFQS WSHPQFEKGG GSGGGS GGG SWSHPQFEK

UniProtKB: Separin, Separin

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMHepes2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
150.0 mMKClPotassium chloride
0.5 mMTCEPtris(2-carboxyethyl)phosphine
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 1
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 16 / Number real images: 57012 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 106898589 / Details: The particles were automatically selected
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.0) / Software - details: Non-uniform refinement was used / Number images used: 614186
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4.4.0)

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Atomic model buiding 1

Initial modelPDB ID:

7nj1


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9hn0:
Cryo-EM structure of human separase bound to SCC1 (310-550 aa)

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