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- EMDB-2173: Negative stain electron microscopy of a CSN-SCF~Nedd8/Skp2/Cks1 c... -

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Basic information

Entry
Database: EMDB / ID: EMD-2173
TitleNegative stain electron microscopy of a CSN-SCF~Nedd8/Skp2/Cks1 complex
Map data-
Sample
  • Sample: CSN-SCF~Nedd8/Skp2/Cks1
  • Protein or peptide: x 14 types
KeywordsCop9 Signalosome / Cullin-RING Ligases / SCF
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsEnchev RI / Scott DC / da Fonseca P / Schreiber A / Monda JK / Schulman BA / Peter M / Morris EP
CitationJournal: Cell Rep / Year: 2012
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
DepositionAug 11, 2012-
Header (metadata) releaseOct 3, 2012-
Map releaseOct 3, 2012-
UpdateOct 3, 2012-
Current statusOct 3, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2173.jpg

Downloads & links

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Map

FileDownload / File: emd_2173.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation-
Voxel sizeX=Y=Z: 3.47 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-14.358153339999999 - 18.373920439999999
Average (Standard dev.)0.21251245 (±2.6079669)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-29-30-30
Dimensions606060
Spacing606060
CellA=B=C: 208.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.473.473.47
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z208.200208.200208.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-30-29-30
NC/NR/NS606060
D min/max/mean-14.35818.3740.213

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Supplemental data

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Sample components

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Entire : CSN-SCF~Nedd8/Skp2/Cks1

EntireName: CSN-SCF~Nedd8/Skp2/Cks1
Components
  • Sample: CSN-SCF~Nedd8/Skp2/Cks1
  • Protein or peptide: Csn1
  • Protein or peptide: Csn2
  • Protein or peptide: Csn3
  • Protein or peptide: Csn4
  • Protein or peptide: Csn5
  • Protein or peptide: Csn6
  • Protein or peptide: Csn7b
  • Protein or peptide: Csn8
  • Protein or peptide: Cul1
  • Protein or peptide: Rbx1
  • Protein or peptide: Skp1S-phase kinase-associated protein 1
  • Protein or peptide: Skp2
  • Protein or peptide: Cks1
  • Protein or peptide: Nedd8

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Supramolecule #1000: CSN-SCF~Nedd8/Skp2/Cks1

SupramoleculeName: CSN-SCF~Nedd8/Skp2/Cks1 / type: sample / ID: 1000
Details: The sample was purified over affinity pull-down and Superose6 size exclusion chromatography
Oligomeric state: one CSN monomer and one SCF~Nedd8/Skp2/Cks1 monomer
Number unique components: 14
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Csn1

MacromoleculeName: Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 53 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #2: Csn2

MacromoleculeName: Csn2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 52 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #3: Csn3

MacromoleculeName: Csn3 / type: protein_or_peptide / ID: 3 / Details: N-terminal StrepII2x tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 48 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #4: Csn4

MacromoleculeName: Csn4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 46 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #5: Csn5

MacromoleculeName: Csn5 / type: protein_or_peptide / ID: 5 / Details: N-terminal His6 tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #6: Csn6

MacromoleculeName: Csn6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 36 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #7: Csn7b

MacromoleculeName: Csn7b / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 30 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #8: Csn8

MacromoleculeName: Csn8 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 23 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #9: Cul1

MacromoleculeName: Cul1 / type: protein_or_peptide / ID: 9 / Details: N-terminal StrepII2x tag / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 90 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #10: Rbx1

MacromoleculeName: Rbx1 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 12 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: recombinant baculoviru/ MultiBac

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Macromolecule #11: Skp1

MacromoleculeName: Skp1 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 19 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX

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Macromolecule #12: Skp2

MacromoleculeName: Skp2 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 47 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX

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Macromolecule #13: Cks1

MacromoleculeName: Cks1 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX

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Macromolecule #14: Nedd8

MacromoleculeName: Nedd8 / type: protein_or_peptide / ID: 14 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 9 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX

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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

Concentration0.2 mg/mL
BufferpH: 7.8
Details: 15 mM HEPES pH 7.8, 150 mM NaCl, 1% glycerol and 1 mM DTT
StainingType: 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.
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 magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJan 10, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 100 e/Å2 / Details: data was collected on a CCD camera
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, in, house / Number images used: 8055
Detailssee publication

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Atomic model buiding 1

Initial modelPDB ID:

1ldk

SoftwareName: Chimera, Uro
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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