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- PDB-7nj1: CryoEM structure of the human Separase-Securin complex -

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Basic information

Entry
Database: PDB / ID: 7nj1
TitleCryoEM structure of the human Separase-Securin complex
Components
  • Securin
  • Separin
KeywordsHYDROLASE / pseudosubstrate HEAT repeat caspase cell cycle
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 sister chromatid segregation / mitotic cytokinesis ...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 sister chromatid segregation / mitotic cytokinesis / catalytic activity / cysteine-type endopeptidase inhibitor activity / chromosome organization / cysteine-type peptidase activity / molecular function activator activity / APC/C:Cdc20 mediated degradation of Securin / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / SH3 domain binding / mitotic spindle / Separation of Sister Chromatids / spermatogenesis / cell division / cysteine-type endopeptidase activity / DNA repair / apoptotic process / centrosome / proteolysis / nucleus / cytoplasm / cytosol
Similarity search - Function
Securin sister-chromatid separation inhibitor / Securin sister-chromatid separation inhibitor / 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: 2.9 Å
AuthorsYu, J. / Raia, P. / Ghent, C.M. / Raisch, T. / Sadian, Y. / Barford, D. / Raunser, S. / Morgan, D.O. / Boland, A.
Funding support Switzerland, United States, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_185235 Switzerland
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM118053 United States
CitationJournal: 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
DepositionFeb 14, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 4, 2021Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 3 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 3 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Additional map / Part number: 3 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 4, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Aug 18, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update
Revision 2.0Oct 1, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / em_admin ...atom_site / em_admin / em_imaging / em_software / entity_src_gen / pdbx_contact_author / pdbx_poly_seq_scheme / pdbx_struct_oper_list / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / refine_ls_restr / software / struct_conf
Item: _em_admin.last_update / _em_imaging.microscope_model ..._em_admin.last_update / _em_imaging.microscope_model / _entity_src_gen.gene_src_common_name / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_struct_oper_list.symmetry_operation / _pdbx_unobs_or_zero_occ_residues.auth_asym_id / _pdbx_unobs_or_zero_occ_residues.auth_comp_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_unobs_or_zero_occ_residues.label_asym_id / _pdbx_unobs_or_zero_occ_residues.label_comp_id / _pdbx_unobs_or_zero_occ_residues.label_seq_id / _struct_conf.beg_auth_asym_id / _struct_conf.beg_auth_comp_id / _struct_conf.beg_auth_seq_id / _struct_conf.beg_label_asym_id / _struct_conf.beg_label_comp_id / _struct_conf.beg_label_seq_id / _struct_conf.end_auth_asym_id / _struct_conf.end_auth_comp_id / _struct_conf.end_auth_seq_id / _struct_conf.end_label_asym_id / _struct_conf.end_label_comp_id / _struct_conf.end_label_seq_id / _struct_conf.pdbx_PDB_helix_class / _struct_conf.pdbx_PDB_helix_length
Description: Model completeness
Details: We have got a 3.45 A-resolution map of the N-terminal Heat-repeat domain of separase, we then revisited the securin-separase structure (this entry) and found the N-terminal domain in the ...Details: We have got a 3.45 A-resolution map of the N-terminal Heat-repeat domain of separase, we then revisited the securin-separase structure (this entry) and found the N-terminal domain in the model could be improved. With the guidance of high-resolution map of separase and Alphafold3-predicted structure of full-length securin-separase complex, we re-built the N-terminal domain of separase (aa 248-442) and securin (aa 154-163).
Provider: author / Type: Coordinate replacement
Revision 1.1Oct 1, 2025Data content type: EM metadata
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata
Group: Data collection / Data processing ...Data collection / Data processing / Experimental summary / Source and taxonomy
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata
Category: em_admin / em_imaging ...em_admin / em_imaging / em_software / entity_src_gen
Data content type: EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _em_imaging.microscope_model / _entity_src_gen.gene_src_common_name

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Structure visualization

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Assembly

Deposited unit
A: Separin
B: Securin


Theoretical massNumber of molelcules
Total (without water)259,6632
Polymers259,6632
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6940 Å2
ΔGint-39 kcal/mol
Surface area64110 Å2
MethodPISA

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Components

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


Mass: 237610.469 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q14674, separase
#2: Protein Securin / Esp1-associated protein / Pituitary tumor-transforming gene 1 protein / Tumor-transforming protein 1 / hPTTG


Mass: 22052.340 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PTTG1, EAP1, PTTG, TUTR1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O95997
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: Inhibitory complex of human separase bound to securin.
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.8
SpecimenConc.: 0.025 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was monodisperse. We use graphene oxide-coated EM grids.
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: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Calibrated defocus min: 1300 nm / Calibrated defocus max: 2500 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 67 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 4 / Num. of real images: 16540

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Processing

EM software
IDNameCategory
2Topazparticle selection
3EPUimage acquisition
5CTFFINDCTF correction
6GctfCTF correction
7cryoSPARCCTF correction
15RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 205300 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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