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- PDB-9hn4: Cryo-EM structure of human separase bound to phosphorylated SCC1 ... -

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Basic information

Entry
Database: PDB / ID: 9hn4
TitleCryo-EM structure of human separase bound to phosphorylated SCC1 (310-550 aa)
Components
  • Double-strand-break repair protein rad21 homolog
  • Separin
KeywordsCELL CYCLE / Separase / SCC1 / RAD21 / protease / chromosome segregation / Auto-cleavage / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 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 10, 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

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Assembly

Deposited unit
A: Double-strand-break repair protein rad21 homolog
B: Separin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)265,9253
Polymers265,8602
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 Double-strand-break repair protein rad21 homolog / hHR21 / Nuclear matrix protein 1 / NXP-1 / SCC1 homolog


Mass: 27773.189 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD21, HR21, KIAA0078, NXP1, SCC1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O60216
#2: Protein Separin / Caspase-like protein ESPL1 / Extra spindle poles-like 1 protein / Separase


Mass: 238086.344 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
#3: 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: Human separase bound to phosphorylated SCC1 (310-550 aa)
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.262 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
280 mMPotassium chlorideKCl1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 0.06 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: 130000 X / Nominal defocus max: 2000 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: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7451
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
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: 6692171 / Details: The particles were automatically selected
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 298574 / 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: 2.93 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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