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- EMDB-2569: Electron microscopy of the native human COP9 signalosome -

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Basic information

Entry
Database: EMDB / ID: EMD-2569
TitleElectron microscopy of the native human COP9 signalosome
Map datanegative stain reconstruction of native human CSN
Sample
  • Sample: CSN from human red blood cells
  • Protein or peptide: COP9 signalosome
KeywordsCSN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsRockel B / Schmaler T / Dubiel W
CitationJournal: Biochem Biophys Res Commun / Year: 2014
Title: Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes.
Authors: Beate Rockel / Tilo Schmaler / Xiaohua Huang / Wolfgang Dubiel /
Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN ...The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes.
History
DepositionFeb 4, 2014-
Header (metadata) releaseFeb 12, 2014-
Map releaseNov 19, 2014-
UpdateNov 19, 2014-
Current statusNov 19, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2569.jpg

Downloads & links

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Map

FileDownload / File: emd_2569.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnegative stain reconstruction of native human CSN
Voxel sizeX=Y=Z: 3.56 Å
Density
Contour LevelBy AUTHOR: 0.017 / Movie #1: 0.017
Minimum - Maximum-0.05361367 - 0.11952274
Average (Standard dev.)0.00109329 (±0.0096371)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 227.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.563.563.56
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z227.840227.840227.840
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-0.0540.1200.001

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Supplemental data

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Sample components

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Entire : CSN from human red blood cells

EntireName: CSN from human red blood cells
Components
  • Sample: CSN from human red blood cells
  • Protein or peptide: COP9 signalosome

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Supramolecule #1000: CSN from human red blood cells

SupramoleculeName: CSN from human red blood cells / type: sample / ID: 1000 / Number unique components: 1

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Macromolecule #1: COP9 signalosome

MacromoleculeName: COP9 signalosome / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human)

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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

StainingType: NEGATIVE / Details: uranyl acetate
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
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJan 20, 2012
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k)
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: xmipp / Number images used: 61236

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