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- EMDB-3384: A closed conformation of the C. elegans separase-securin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3384
TitleA closed conformation of the C. elegans separase-securin complex
Map dataReconstruction of C. elegans separase-securinn complex
Sample
  • Sample: separase-securin complex
  • Protein or peptide: separase
  • Protein or peptide: securin
Keywordschromosome segregation / protease
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)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsBachmann G / Morris E / Bayliss R
CitationJournal: Open Biol / Year: 2016
Title: A closed conformation of the Caenorhabditis elegans separase-securin complex.
Authors: Gudrun Bachmann / Mark W Richards / Anja Winter / Fabienne Beuron / Edward Morris / Richard Bayliss /
Abstract: The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory ...The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase-securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21-24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase-securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change.
History
DepositionMar 14, 2016-
Header (metadata) releaseMay 4, 2016-
Map releaseJun 15, 2016-
UpdateAug 24, 2016-
Current statusAug 24, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

3384.png

Downloads & links

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Map

FileDownload / File: emd_3384.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of C. elegans separase-securinn complex
Voxel sizeX=Y=Z: 2.915 Å
Density
Contour LevelBy AUTHOR: 4.0 / Movie #1: 4
Minimum - Maximum-7.97223091 - 18.95828247
Average (Standard dev.)-0.00000001 (±1.00000012)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions100100100
Spacing100100100
CellA=B=C: 291.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.9152.9152.915
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z291.500291.500291.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-50-50-50
NC/NR/NS100100100
D min/max/mean-7.97218.958-0.000

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Supplemental data

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Sample components

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Entire : separase-securin complex

EntireName: separase-securin complex
Components
  • Sample: separase-securin complex
  • Protein or peptide: separase
  • Protein or peptide: securin

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Supramolecule #1000: separase-securin complex

SupramoleculeName: separase-securin complex / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: one monomer of separase bound to one monomer of securin
Number unique components: 2
Molecular weightExperimental: 174 KDa / Theoretical: 171 KDa / Method: Multi-angle light scattering

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Macromolecule #1: separase

MacromoleculeName: separase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: momomer / Recombinant expression: Yes
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Molecular weightTheoretical: 144 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceUniProtKB: Separin homolog sep-1

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Macromolecule #2: securin

MacromoleculeName: securin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Molecular weightTheoretical: 27 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceUniProtKB: Securin-like protein

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7 / Details: 50mM Tris, 0.5M NaCl, 2mM beta-mercaptoethanol
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 20 seconds.
GridDetails: Thin carbon support on Quantifoil R 1.2/1.3 grids glow discharged in residual air
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.85 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateNov 10, 2010
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Sampling interval: 15 µm / Average electron dose: 100 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: Imagic, Spider / Number images used: 5764

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