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- PDB-9hma: Cryo-EM structure of apo human separase -

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Basic information

Entry
Database: PDB / ID: 9hma
TitleCryo-EM structure of apo human separase
ComponentsSeparin
KeywordsCELL CYCLE / Separase / SCC1 / RAD21 / protease / chromosome segregation / Auto-cleavage
Function / homology
Function and homology information


negative regulation of sister chromatid cohesion / separase / meiotic chromosome separation / mitotic sister chromatid separation / homologous chromosome segregation / establishment of mitotic spindle localization / meiotic spindle organization / positive regulation of mitotic metaphase/anaphase transition / mitotic cytokinesis / catalytic activity ...negative regulation of sister chromatid cohesion / separase / meiotic chromosome separation / mitotic sister chromatid separation / homologous chromosome segregation / establishment of mitotic spindle localization / meiotic spindle organization / positive regulation of mitotic metaphase/anaphase transition / mitotic cytokinesis / catalytic activity / mitotic sister chromatid segregation / cysteine-type peptidase activity / mitotic spindle / Separation of Sister Chromatids / cysteine-type endopeptidase activity / apoptotic process / centrosome / proteolysis / nucleus / cytoplasm / cytosol
Similarity search - Function
Peptidase C50, separase / SEPARIN core domain / Separin, protease domain / SEPARIN core domain profile.
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYu, J. / Schmidt, S. / Botto, M. / Boland, A.
Funding support Switzerland, 2items
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 7, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Separin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)243,2172
Polymers243,1511
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Separin / Caspase-like protein ESPL1 / Extra spindle poles-like 1 protein / Separase


Mass: 243151.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ESPL1, ESP1, KIAA0165 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14674, separase
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
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: Apo human separase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.23 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHepes1
2100 mMPotassium chlorideKCl1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3314
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.4.0particle selection
2EPU2.13image acquisition
4cryoSPARC4.4.0CTF correction
7UCSF ChimeraX1.8model fitting
9PHENIX1.20.1_4487model refinement
10Coot0.9.8.92model refinement
11cryoSPARC4.4.0initial Euler assignment
12cryoSPARC4.4.0final Euler assignment
13cryoSPARC4.4.0classification
14cryoSPARC4.4.03D reconstructionNon-uniform refinement was used
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4023196 / Details: The particles were automatically selected
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 224027 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7NJ1

7nj1


Pdb chain-ID: A / Accession code: 7NJ1 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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