+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3401 | |||||||||
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Title | Electron cryo-microscopy of CSN-SCF-N8 complex | |||||||||
Map data | reconstruction of CSN5H138A-N8_SCF/Skp2 | |||||||||
Sample |
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Keywords | COP9 signalosome / Cullin-RING ligase / SCF / deneddylation | |||||||||
Function / homology | Function and homology information 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 / eukaryotic translation initiation factor 3 complex / regulation of protein neddylation / protein deneddylation / cellular response to chemical stress / cullin-RING-type E3 NEDD8 transferase / PcG protein complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / Cul7-RING ubiquitin ligase complex / mitotic cell cycle phase transition / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / COP9 signalosome / regulation of proteolysis / metal-dependent deubiquitinase activity / activation of NF-kappaB-inducing kinase activity / positive regulation of ubiquitin protein ligase activity / maintenance of protein location in nucleus / positive regulation of protein autoubiquitination / protein neddylation / cyclin-dependent protein serine/threonine kinase activator activity / NEDD8 ligase activity / Hydrolases; Acting on peptide bonds (peptidases) / Cul5-RING ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / ubiquitin-ubiquitin ligase activity / Cul4A-RING E3 ubiquitin ligase complex / RHOBTB1 GTPase cycle / Cul2-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / negative regulation of type I interferon production / ubiquitin ligase complex scaffold activity / inner cell mass cell proliferation / positive regulation of intracellular estrogen receptor signaling pathway / Cul3-RING ubiquitin ligase complex / protein monoubiquitination / Prolactin receptor signaling / cullin family protein binding / protein deubiquitination / skeletal muscle cell differentiation / TGF-beta receptor signaling activates SMADs / regulation of mitotic cell cycle / ubiquitin ligase-substrate adaptor activity / regulation of JNK cascade / response to light stimulus / protein K48-linked ubiquitination / cyclin-dependent protein kinase holoenzyme complex / anatomical structure morphogenesis / Nuclear events stimulated by ALK signaling in cancer / JNK cascade / positive regulation of TORC1 signaling / translation initiation factor activity / Regulation of BACH1 activity / T cell activation / post-translational protein modification / MAP3K8 (TPL2)-dependent MAPK1/3 activation / intrinsic apoptotic signaling pathway / ubiquitin binding / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / molecular function activator activity / Degradation of DVL / Vpu mediated degradation of CD4 / Recognition of DNA damage by PCNA-containing replication complex / Dectin-1 mediated noncanonical NF-kB signaling / cellular response to amino acid stimulus / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / Negative regulation of NOTCH4 signaling / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Iron uptake and transport / DNA Damage Recognition in GG-NER / animal organ morphogenesis / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / G1/S transition of mitotic cell cycle / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / negative regulation of canonical Wnt signaling pathway / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / RING-type E3 ubiquitin transferase / protein modification process / modification-dependent protein catabolic process / Degradation of beta-catenin by the destruction complex / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Dual Incision in GG-NER Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.2 Å | |||||||||
Authors | Mosadeghi R / Reichermeier KM / Winkler M / Schreiber A / Reitsma JM / Zhang Y / Stengel F / Cao J / Kim M / Sweredoski MJ ...Mosadeghi R / Reichermeier KM / Winkler M / Schreiber A / Reitsma JM / Zhang Y / Stengel F / Cao J / Kim M / Sweredoski MJ / Hess S / Leitner A / Aebersold R / Peter M / Deshaies RJ / Enchev RI | |||||||||
Citation | 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3401.map.gz | 37.4 MB | EMDB map data format | |
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Header (meta data) | emd-3401-v30.xml emd-3401.xml | 29.3 KB 29.3 KB | Display Display | EMDB header |
Images | EMD-3401_image.tif | 732.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3401 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3401 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3401.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | reconstruction of CSN5H138A-N8_SCF/Skp2 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.74 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
+Entire : CSN5H138A-N8_SCF/Skp2
+Supramolecule #1000: CSN5H138A-N8_SCF/Skp2
+Macromolecule #1: Csn1
+Macromolecule #2: Csn2
+Macromolecule #3: Csn3
+Macromolecule #4: Csn4
+Macromolecule #5: Csn5
+Macromolecule #6: Csn6
+Macromolecule #7: Csn7b
+Macromolecule #8: Csn8
+Macromolecule #9: Cul1
+Macromolecule #10: Rbx1
+Macromolecule #11: Nedd8
+Macromolecule #12: Skp1
+Macromolecule #13: Skp2
+Macromolecule #14: Cks1
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | 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 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 80460 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 60000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
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. |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: micrographs |
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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 |
Details | 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. |
-Atomic model buiding 1
Initial model | PDB ID: 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 |
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Software | Name: Chmiera |
Details | some domains were fitted separately to account for conformational rearrangements, see publication for details |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap |
-Atomic model buiding 2
Initial model | PDB ID: 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 |
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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 |
-Atomic model buiding 3
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B |
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Software | Name: Chmiera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap |