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- EMDB-1700: Negative stain EM structure of human native COP9 Signalosome (CSN) -

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Basic information

Entry
Database: EMDB / ID: EMD-1700
TitleNegative stain EM structure of human native COP9 Signalosome (CSN)
Map dataApo CSN map
Sample
  • Sample: Human erythrocyte COP9 Signalosome
  • Protein or peptide: Cop9 Signalosome
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsEnchev R / Schreiber A / Beuron F / Morris E
CitationJournal: Structure / Year: 2010
Title: Structural insights into the COP9 signalosome and its common architecture with the 26S proteasome lid and eIF3.
Authors: Radoslav I Enchev / Anne Schreiber / Fabienne Beuron / Edward P Morris /
Abstract: The evolutionary conserved COP9 signalosome (CSN), a large multisubunit complex, plays a central role in regulating ubiquitination and cell signaling. Here we report recombinant insect cell ...The evolutionary conserved COP9 signalosome (CSN), a large multisubunit complex, plays a central role in regulating ubiquitination and cell signaling. Here we report recombinant insect cell expression and two-step purification of human CSN and demonstrate its functional assembly. We further obtain a three-dimensional structure of both native and recombinant CSN using electron microscopy and single particle analysis. Antibody labeling of CSN5 and segmentation of the structure suggest a likely subunit distribution and the architecture of its helical repeat subunits is revealed. We compare the structure of CSN with its homologous complexes, the 26S proteasome lid and eIF3, and propose a conserved architecture implying similar assembly pathways and/or conserved substrate interaction modes.
History
DepositionFeb 8, 2010-
Header (metadata) releaseMay 13, 2010-
Map releaseMay 13, 2010-
UpdateMay 13, 2010-
Current statusMay 13, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1700.jpg

Downloads & links

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Map

FileDownload / File: emd_1700.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationApo CSN map
Voxel sizeX=Y=Z: 3.47 Å
Density
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.00891048 - 0.245599
Average (Standard dev.)0.000351193 (±0.00567995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-63-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 444.16 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.473.473.47
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z444.160444.160444.160
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ121121121
MAP C/R/S123
start NC/NR/NS-64-63-64
NC/NR/NS128128128
D min/max/mean-0.0090.2460.000

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Supplemental data

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Sample components

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Entire : Human erythrocyte COP9 Signalosome

EntireName: Human erythrocyte COP9 Signalosome
Components
  • Sample: Human erythrocyte COP9 Signalosome
  • Protein or peptide: Cop9 Signalosome

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Supramolecule #1000: Human erythrocyte COP9 Signalosome

SupramoleculeName: Human erythrocyte COP9 Signalosome / type: sample / ID: 1000 / Oligomeric state: Heterooctamer / Number unique components: 1
Molecular weightExperimental: 321 KDa / Theoretical: 321 KDa / Method: Native mass spectrometry

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Macromolecule #1: Cop9 Signalosome

MacromoleculeName: Cop9 Signalosome / type: protein_or_peptide / ID: 1 / Name.synonym: CSN / Number of copies: 1 / Oligomeric state: Octamer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: Erythrocytes / Location in cell: Nucleus

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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: 7.8
Details: 15 mM HEPES pH 7.8, 200 mM NaCl, 1% glycerol and 2 mM DTT
StainingType: NEGATIVE
Details: Sample was applied to glow-discharged quantifoil grids coated with thin carbon, washed with buffer and stained with 2% uranyl acetate
GridDetails: Quantifoil 400 mesh R2/2
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 / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Digitization - Sampling interval: 3.47 µm / Average electron dose: 100 e/Å2
Details: Initial selection was made manually from more than 100 2048x2048 CCD frames
Tilt angle min0
Tilt angle max0
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: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, SPIDER, EMAN / Number images used: 7877

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