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- PDB-5mz6: Cryo-EM structure of a Separase-Securin complex from Caenorhabdit... -

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Basic information

Entry
Database: PDB / ID: 5mz6
TitleCryo-EM structure of a Separase-Securin complex from Caenorhabditis elegans at 3.8 A resolution
Components
  • Interactor of FizzY protein
  • SEParase
KeywordsCELL CYCLE / caspase / cohesin / cleavage
Function / homology
Function and homology information


Separation of Sister Chromatids / separase-securin complex / eggshell formation / metaphase plate / regulation of nematode larval development / separase / cortical granule exocytosis / maintenance of meiotic sister chromatid cohesion / meiotic chromosome separation / maintenance of mitotic sister chromatid cohesion ...Separation of Sister Chromatids / separase-securin complex / eggshell formation / metaphase plate / regulation of nematode larval development / separase / cortical granule exocytosis / maintenance of meiotic sister chromatid cohesion / meiotic chromosome separation / maintenance of mitotic sister chromatid cohesion / polarity specification of anterior/posterior axis / polar body extrusion after meiotic divisions / cortical granule / regulation of centriole-centriole cohesion / mitotic sister chromatid separation / regulation of exocytosis / multicellular organismal reproductive process / meiotic spindle / regulation of locomotion / centrosome localization / cleavage furrow / centrosome duplication / mitotic cytokinesis / condensed chromosome / condensed nuclear chromosome / spindle microtubule / protein localization / spindle / mitotic spindle / protein processing / nuclear envelope / chromosome / cell cortex / midbody / protease binding / protein stabilization / cysteine-type endopeptidase activity / centrosome / ubiquitin protein ligase binding / nucleus / cytoplasm
Similarity search - Function
Peptidase family C50 / Peptidase C50, separase / SEPARIN core domain / SEPARIN core domain profile.
Similarity search - Domain/homology
Separin homolog sep-1 / Securin-like protein
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsBoland, A. / Martin, T.G. / Zhang, Z. / Yang, J. / Bai, X.C. / Chang, L. / Scheres, S.H.W. / Barford, D.
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution.
Authors: Andreas Boland / Thomas G Martin / Ziguo Zhang / Jing Yang / Xiao-Chen Bai / Leifu Chang / Sjors H W Scheres / David Barford /
Abstract: Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle ...Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle function through cleavage of kendrin and Slk19. To understand the mechanisms of securin regulation of separase, we used single-particle cryo-electron microscopy (cryo-EM) to determine a near-atomic-resolution structure of the Caenorhabditis elegans separase-securin complex. Separase adopts a triangular-shaped bilobal architecture comprising an N-terminal tetratricopeptide repeat (TPR)-like α-solenoid domain docked onto the conserved C-terminal protease domain. Securin engages separase in an extended antiparallel conformation, interacting with both lobes. It inhibits separase by interacting with the catalytic site through a pseudosubstrate mechanism, thus revealing that in the inhibited separase-securin complex, the catalytic site adopts a conformation compatible with substrate binding. Securin is protected from cleavage because an aliphatic side chain at the P1 position represses protease activity by disrupting the organization of catalytic site residues.
History
DepositionJan 31, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 8, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2017Group: Database references
Revision 1.2Apr 19, 2017Group: Database references
Revision 1.3Aug 30, 2017Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
1: SEParase
B: Interactor of FizzY protein


Theoretical massNumber of molelcules
Total (without water)171,3442
Polymers171,3442
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5510 Å2
ΔGint-29 kcal/mol
Surface area39900 Å2
MethodPISA

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Components

#1: Protein SEParase / / Separase


Mass: 144315.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: sep-1, CELE_Y47G6A.12, Y47G6A.12 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: G5ED39
#2: Protein Interactor of FizzY protein


Mass: 27027.646 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: ify-1, C27A2.3, CELE_C27A2.3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q18235

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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: Caenorhabditis elegans Separase-Securin complex / Type: COMPLEX
Details: C. elegans Separase-Securin complex at 3.8 Angstrom resolution refined with Relion.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: NO
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.8
SpecimenConc.: 0.02 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample
Specimen supportDetails: Glow discharging was performed before applying graphene oxide solution onto the grid. Grid was washed three times, dried and sample was applied. For detailed information see: https: ...Details: Glow discharging was performed before applying graphene oxide solution onto the grid. Grid was washed three times, dried and sample was applied. For detailed information see: https://figshare.com/articles/Graphene_Oxide_Grid_Preparation/3178669
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Custom Manual Plunger

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.8 sec. / Electron dose: 1.25 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: -20 eV
Image scansWidth: 3710 / Height: 3710

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
4GctfCTF correction
7Coot0.88model fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
12RELION23D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name: 1 / Space group num: 1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103696 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058569
ELECTRON MICROSCOPYf_angle_d0.89111591
ELECTRON MICROSCOPYf_dihedral_angle_d11.8255189
ELECTRON MICROSCOPYf_chiral_restr0.0491312
ELECTRON MICROSCOPYf_plane_restr0.0061473

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