regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / COP9 signalosome assembly / trophectodermal cell proliferation / macrophage migration inhibitory factor binding / positive regulation of protein polyubiquitination / regulation of IRE1-mediated unfolded protein response / Parkin-FBXW7-Cul1 ubiquitin ligase complex / exosomal secretion / deNEDDylase activity / GTPase inhibitor activity ...regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / COP9 signalosome assembly / trophectodermal cell proliferation / macrophage migration inhibitory factor binding / positive regulation of protein polyubiquitination / regulation of IRE1-mediated unfolded protein response / Parkin-FBXW7-Cul1 ubiquitin ligase complex / exosomal secretion / deNEDDylase activity / GTPase inhibitor activity / F-box domain binding / regulation of protein neddylation / eukaryotic translation initiation factor 3 complex / protein deneddylation / Aberrant regulation of mitotic exit in cancer due to RB1 defects / PcG protein complex / cullin-RING-type E3 NEDD8 transferase / cellular response to chemical stress / NEDD8 transferase activity / mitotic cell cycle phase transition / cullin-RING ubiquitin ligase complex / COP9 signalosome / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / activation of NF-kappaB-inducing kinase activity / positive regulation of ubiquitin protein ligase activity / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / regulation of proteolysis / maintenance of protein location in nucleus / positive regulation of protein autoubiquitination / protein neddylation / metal-dependent deubiquitinase activity / cyclin-dependent protein serine/threonine kinase activator activity / Hydrolases; Acting on peptide bonds (peptidases) / NEDD8 ligase activity / RHOBTB1 GTPase cycle / Cul5-RING ubiquitin ligase complex / negative regulation of response to oxidative stress / positive regulation of intracellular estrogen receptor signaling pathway / ubiquitin-ubiquitin ligase activity / Cul4A-RING E3 ubiquitin ligase complex / SCF ubiquitin ligase complex / inner cell mass cell proliferation / Cul2-RING ubiquitin ligase complex / negative regulation of type I interferon production / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / protein deubiquitination / Prolactin receptor signaling / skeletal muscle cell differentiation / TGF-beta receptor signaling activates SMADs / protein monoubiquitination / cullin family protein binding / protein K63-linked ubiquitination / regulation of JNK cascade / ubiquitin-like ligase-substrate adaptor activity / positive regulation of double-strand break repair via homologous recombination / response to light stimulus / cyclin-dependent protein kinase holoenzyme complex / anatomical structure morphogenesis / Nuclear events stimulated by ALK signaling in cancer / protein K48-linked ubiquitination / JNK cascade / positive regulation of TORC1 signaling / regulation of mitotic cell cycle / T cell activation / translation initiation factor activity / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / intrinsic apoptotic signaling pathway / post-translational protein modification / ubiquitin binding / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / molecular function activator activity / Vpu mediated degradation of CD4 / Degradation of DVL / Dectin-1 mediated noncanonical NF-kB signaling / Recognition of DNA damage by PCNA-containing replication complex / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / Negative regulation of NOTCH4 signaling / Iron uptake and transport / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / animal organ morphogenesis / cellular response to amino acid stimulus / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / DNA Damage Recognition in GG-NER / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / RING-type E3 ubiquitin transferase / Degradation of beta-catenin by the destruction complex / protein modification process / negative regulation of canonical Wnt signaling pathway / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha Similarity search - Function
Journal: Elife / Year: 2016 Title: Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle. Authors: Ruzbeh Mosadeghi / Kurt M Reichermeier / Martin Winkler / Anne Schreiber / Justin M Reitsma / Yaru Zhang / Florian Stengel / Junyue Cao / Minsoo Kim / Michael J Sweredoski / Sonja Hess / ...Authors: Ruzbeh Mosadeghi / Kurt M Reichermeier / Martin Winkler / Anne Schreiber / Justin M Reitsma / Yaru Zhang / Florian Stengel / Junyue Cao / Minsoo Kim / Michael J Sweredoski / Sonja Hess / Alexander Leitner / Ruedi Aebersold / Matthias Peter / Raymond J Deshaies / Radoslav I Enchev / Abstract: The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes ...The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.
History
Deposition
Mar 29, 2016
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Header (metadata) release
Apr 6, 2016
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Map release
Apr 6, 2016
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Update
Apr 13, 2016
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Current status
Apr 13, 2016
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_3401.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
reconstruction of CSN5H138A-N8_SCF/Skp2
Voxel size
X=Y=Z: 1.74 Å
Density
Contour Level
By AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum
-0.08129014 - 0.22193521
Average (Standard dev.)
0.00045421 (±0.01186982)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
220
220
220
Spacing
220
220
220
Cell
A=B=C: 382.8 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.74
1.74
1.74
M x/y/z
220
220
220
origin x/y/z
0.000
0.000
0.000
length x/y/z
382.800
382.800
382.800
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-30
0
-64
NX/NY/NZ
61
61
129
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
220
220
220
D min/max/mean
-0.081
0.222
0.000
-
Supplemental data
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Sample components
+
Entire : CSN5H138A-N8_SCF/Skp2
Entire
Name: CSN5H138A-N8_SCF/Skp2
Components
Sample: CSN5H138A-N8_SCF/Skp2
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: Nedd8
Protein or peptide: Skp1
Protein or peptide: Skp2
Protein or peptide: Cks1
+
Supramolecule #1000: CSN5H138A-N8_SCF/Skp2
Supramolecule
Name: CSN5H138A-N8_SCF/Skp2 / type: sample / ID: 1000 / Oligomeric state: one CSN monomer and one SCF monomer / Number unique components: 14
Molecular weight
Theoretical: 500 KDa
+
Macromolecule #1: Csn1
Macromolecule
Name: Csn1 / type: protein_or_peptide / ID: 1 / Name.synonym: Cops1 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 55 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 1
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Macromolecule #2: Csn2
Macromolecule
Name: Csn2 / type: protein_or_peptide / ID: 2 / Name.synonym: Cops2 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 51 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 2
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Macromolecule #3: Csn3
Macromolecule
Name: Csn3 / type: protein_or_peptide / ID: 3 / Name.synonym: Cops3 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 47 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 3
+
Macromolecule #4: Csn4
Macromolecule
Name: Csn4 / type: protein_or_peptide / ID: 4 / Name.synonym: Cops4 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 46 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 4
+
Macromolecule #5: Csn5
Macromolecule
Name: Csn5 / type: protein_or_peptide / ID: 5 / Name.synonym: Cops5 / Details: His6 tag on the N-terminus / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 37 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 5
+
Macromolecule #6: Csn6
Macromolecule
Name: Csn6 / type: protein_or_peptide / ID: 6 / Name.synonym: Cops6 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 36 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 6
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Macromolecule #7: Csn7b
Macromolecule
Name: Csn7b / type: protein_or_peptide / ID: 7 / Name.synonym: Cops7b / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 29.6 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 7b
+
Macromolecule #8: Csn8
Macromolecule
Name: Csn8 / type: protein_or_peptide / ID: 8 / Name.synonym: Cops8 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 23 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: COP9 signalosome complex subunit 8
+
Macromolecule #9: Cul1
Macromolecule
Name: Cul1 / type: protein_or_peptide / ID: 9 / Details: StrepII2x tag on the N-terminus / Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 89 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: Cullin-1
+
Macromolecule #10: Rbx1
Macromolecule
Name: Rbx1 / type: protein_or_peptide / ID: 10 / Name.synonym: Roc1 / Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 12 KDa
Recombinant expression
Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
Sequence
UniProtKB: E3 ubiquitin-protein ligase RBX1
+
Macromolecule #11: Nedd8
Macromolecule
Name: Nedd8 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 9 KDa
Recombinant expression
Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
Sequence
UniProtKB: NEDD8
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Macromolecule #12: Skp1
Macromolecule
Name: Skp1 / type: protein_or_peptide / ID: 12 Details: see Enchev et al 2012 Cell Reports for details of the used construct Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Recombinant expression
Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
Sequence
UniProtKB: S-phase kinase-associated protein 1
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Macromolecule #13: Skp2
Macromolecule
Name: Skp2 / type: protein_or_peptide / ID: 13 Details: see Enchev et al 2012 Cell Reports for details of the used construct Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Recombinant expression
Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
Sequence
UniProtKB: S-phase kinase-associated protein 2
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Macromolecule #14: Cks1
Macromolecule
Name: Cks1 / type: protein_or_peptide / ID: 14 Details: see Enchev et al 2012 Cell Reports for details of the used construct Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
Source (natural)
Organism: Homo sapiens (human) / synonym: Human
Molecular weight
Theoretical: 9.6 KDa
Recombinant expression
Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
pH: 7.6 / Details: 15 mM HEPES, pH 7.6, 100 mM NaCl, 0.5 mM DTT
Grid
Details: Quantifoil grids (R1.2/1.3 Cu 400 mesh), freshly coated with an extra layer of thin carbon and glow-discharged for 2 min at 50 mA and 0.2 mbar vacuum
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: HOMEMADE PLUNGER Method: the sample was diluted to 0.1 mg/ml and 2 ul were applied to Quantifoil grids (R1.2/1.3 Cu 400 mesh), freshly coated with an extra layer of thin carbon and glow-discharged for 2 min at 50 mA ...Method: the sample was diluted to 0.1 mg/ml and 2 ul were applied to Quantifoil grids (R1.2/1.3 Cu 400 mesh), freshly coated with an extra layer of thin carbon and glow-discharged for 2 min at 50 mA and 0.2 mbar vacuum. The grids were manually blotted to produce a thin sample film and plunge-frozen into liquid ethane
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Electron microscopy
Microscope
FEI TITAN KRIOS
Temperature
Min: 90 K / Max: 110 K / Average: 100 K
Alignment procedure
Legacy - Astigmatism: Objective lens astigmatism was corrected manually at a nominal magnification of 100,000x
Date
May 12, 2015
Image recording
Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 25 e/Å2 Details: Microgrpahs were the average of six frames recorded by the direct electron detector. All micrographs were initially visually inspected and only those with appropriate ice thickness as well ...Details: Microgrpahs were the average of six frames recorded by the direct electron detector. All micrographs were initially visually inspected and only those with appropriate ice thickness as well as Thon rings in their power spectra showing regularity and extending to 6 A or beyond were used for subsequent analysis.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
CTF-estimation and subsequent correction were performed using RELION (Scheres, 2012) and CTFFIND3 (Mindell and Grigorieff, 2003). All micrographs were initially visually inspected and only those with appropriate ice thickness as well as Thon rings in their power spectra showing regularity and extending to 6 A or beyond were used for subsequent analysis. In order to generate 2D references for automated particle selection, ~ 4,000 single particles were manually picked and subjected to 2D classification in RELION. Six well-defined 2D class averages were selected, low-pass filtered to 35 A to prevent reference bias, and used as references. Approximately 150,000 single particles were automatically selected and subjected to reference-free 2D and 3D classification, in order to de-select the particles, which resulted in poorly defined or noisy averages. Approximately half of these single particles resulted in a well-defined 3D class average, which resembled the previously published negative stain EM map of the same complex (Enchev et al., 2012). This dataset was subject to 3D auto-refinement in RELION, using a version low-pass filtered to 50 A as an initial reference. The converged map was further post-processed in RELION, using MTF-correction, FSC-weighting and a soft spherical mask with a 5-pixel fall-off.
CTF correction
Details: micrographs
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: OTHER / Software - Name: relion, CTFFIND3 / Number images used: 75000
Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: H
Software
Name: Chmiera
Details
Csn7b was modeled using Csn7a as a template on the Phyre2 server; some domains were fitted separately to account for conformational rearrangements, see publication for details
Refinement
Space: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap
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