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- PDB-9hn5: Cryo-EM structure of human separase bound to phosphorylated SCC1 ... -

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Basic information

Entry
Database: PDB / ID: 9hn5
TitleCryo-EM structure of human separase bound to phosphorylated SCC1 (100-320 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 / homologous chromosome segregation / negative regulation of glial cell apoptotic process / establishment of mitotic spindle localization / negative regulation of G2/M transition of mitotic cell cycle / meiotic spindle organization / replication-born double-strand break repair via sister chromatid exchange / positive regulation of mitotic metaphase/anaphase transition / establishment of mitotic sister chromatid cohesion / chromatin looping / reciprocal meiotic recombination / sister chromatid cohesion / negative regulation of interleukin-1 beta production / lncRNA binding / mitotic sister chromatid segregation / mitotic cytokinesis / positive regulation of interleukin-10 production / catalytic activity / 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 / midbody / DNA recombination / 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 / centrosome / chromatin binding / regulation of transcription by RNA polymerase II / chromatin / proteolysis / nucleoplasm / nucleus / membrane / cytosol / cytoplasm
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: 2.96 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: To Be Published
Title: Structural insights into cohesin cleavage by human separase
Authors: Yu, J. / Schmidt, S. / Botto, M. / Ghent, C.M. / Morgan, D.O. / Boland, A.
History
DepositionDec 10, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Additional map / Part number: 3 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)263,6663
Polymers263,6012
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: 25514.238 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 / Mutation: C2029S
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 (100-320 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: 1 / Num. of real images: 12867
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
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: 12609660 / Details: The particles were automatically selected
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 195231 / 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: 2.96 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511487
ELECTRON MICROSCOPYf_angle_d0.70515642
ELECTRON MICROSCOPYf_dihedral_angle_d4.1261605
ELECTRON MICROSCOPYf_chiral_restr0.0381852
ELECTRON MICROSCOPYf_plane_restr0.0072008

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