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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2177 | |||||||||
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Title | Negative stain electron microscopy of a CSN complex | |||||||||
![]() | CSN(Csn2dN1-269) | |||||||||
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![]() | Cop9 Signalosome | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 25.0 Å | |||||||||
![]() | Enchev RI / Scott DC / da Fonseca P / Schreiber A / Monda JK / Schulman BA / Peter M / Morris EP | |||||||||
![]() | ![]() Title: Structural basis for a reciprocal regulation between SCF and CSN. Authors: Radoslav I Enchev / Daniel C Scott / Paula C A da Fonseca / Anne Schreiber / Julie K Monda / Brenda A Schulman / Matthias Peter / Edward P Morris / ![]() Abstract: Skp1-Cul1-Fbox (SCF) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 signalosome (CSN). However, CSN also promotes SCF substrate ...Skp1-Cul1-Fbox (SCF) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCF(Skp2/Cks1) and SCF(Fbw7) and found that CSN occludes both SCF functional sites-the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 3.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.9 KB 14.9 KB | Display Display | ![]() |
Images | ![]() | 15.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 188.1 KB | Display | ![]() |
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Full document | ![]() | 187.3 KB | Display | |
Data in XML | ![]() | 5.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | CSN(Csn2dN1-269) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.47 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : CSN(Csn2dN1-269)
Entire | Name: CSN(Csn2dN1-269) |
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Components |
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-Supramolecule #1000: CSN(Csn2dN1-269)
Supramolecule | Name: CSN(Csn2dN1-269) / type: sample / ID: 1000 Details: The sample was purified over affinity pull-down and Superose6 size exclusion chromatography Oligomeric state: one CSN monomer / Number unique components: 8 |
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Molecular weight | Theoretical: 290 KDa |
-Macromolecule #1: Csn1
Macromolecule | Name: Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 53 KDa |
Recombinant expression | Organism: ![]() |
-Macromolecule #2: Csn2
Macromolecule | Name: Csn2 / type: protein_or_peptide / ID: 2 / Details: deleted residues 1-269 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 52 KDa |
Recombinant expression | Organism: ![]() |
-Macromolecule #3: Csn3
Macromolecule | Name: Csn3 / type: protein_or_peptide / ID: 3 / Details: N-terminal StrepII2x tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 48 KDa |
Recombinant expression | Organism: ![]() |
-Macromolecule #4: Csn4
Macromolecule | Name: Csn4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 46 KDa |
Recombinant expression | Organism: ![]() |
-Macromolecule #5: Csn5
Macromolecule | Name: Csn5 / type: protein_or_peptide / ID: 5 / Details: N-terminal His6 tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 40 KDa |
Recombinant expression | Organism: ![]() |
-Macromolecule #6: Csn6
Macromolecule | Name: Csn6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 36 KDa |
Recombinant expression | Organism: ![]() |
-Macromolecule #7: Csn7b
Macromolecule | Name: Csn7b / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 30 KDa |
Recombinant expression | Organism: ![]() |
-Macromolecule #8: Csn8
Macromolecule | Name: Csn8 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 23 KDa |
Recombinant expression | Organism: ![]() |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.2 mg/mL |
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Buffer | pH: 7.8 Details: 15 mM HEPES pH 7.8, 150 mM NaCl, 1% glycerol and 1 mM DTT |
Staining | Type: NEGATIVE Details: Quantifoil grids (R2/2 Cu 400 mesh) coated with thin carbon floated from mica were glow-discharged for 30 seconds at 50 mA and 0.2 mbar vacuum. 3 ul purified samples were applied for 1 min ...Details: Quantifoil grids (R2/2 Cu 400 mesh) coated with thin carbon floated from mica were glow-discharged for 30 seconds at 50 mA and 0.2 mbar vacuum. 3 ul purified samples were applied for 1 min to the grids. Following two brief buffer washes, the grids were stained with 2% uranyl acetate, gently blotted using filter paper and air-dried. |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Date | Jan 10, 2011 |
Image recording | Category: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 100 e/Å2 / Details: data was collected on a CCD camera |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Details | see publication |
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Final reconstruction | Applied 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, in-house / Number images used: 4379 |