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- EMDB-3401: Electron cryo-microscopy of CSN-SCF-N8 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3401
TitleElectron cryo-microscopy of CSN-SCF-N8 complex
Map datareconstruction of CSN5H138A-N8_SCF/Skp2
Sample
  • Sample: CSN5H138A-N8_SCF/Skp2
  • Protein or peptide: x 14 types
KeywordsCOP9 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
COP9 signalosome, subunit CSN8 / COP9 signalosome complex subunit 7, helix I / : / : / COP9 signalosome complex subunit 7a helix I domain / COP9 signalosome complex subunit 1, C-terminal helix / COP9 signalosome complex subunit 3-like, C-terminal helix / COP9 signalosome subunit 6 / Cop9 signalosome subunit 5 C-terminal domain / COP9 signalosome complex subunit 4, helix turn helix domain ...COP9 signalosome, subunit CSN8 / COP9 signalosome complex subunit 7, helix I / : / : / COP9 signalosome complex subunit 7a helix I domain / COP9 signalosome complex subunit 1, C-terminal helix / COP9 signalosome complex subunit 3-like, C-terminal helix / COP9 signalosome subunit 6 / Cop9 signalosome subunit 5 C-terminal domain / COP9 signalosome complex subunit 4, helix turn helix domain / Cop9 signalosome subunit 5 C-terminal domain / CSN4/RPN5/eIF3a helix turn helix domain / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. / Cyclin-dependent kinases regulatory subunits signature 2. / Cyclin-dependent kinase regulatory subunit / Nedd8-like ubiquitin / Eukaryotic translation initiation factor 3 subunit M eIF3m/COP9 signalosome complex subunit 7 COPS7 / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / A Receptor for Ubiquitination Targets / F-box domain profile. / F-box-like domain superfamily / F-box-like / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / F-box domain / 26S proteasome regulatory subunit Rpn7, N-terminal / Cullin protein neddylation domain / Cullin, conserved site / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / Cullin / 26S proteasome subunit RPN7 / Cullin family signature. / 26S Proteasome non-ATPase regulatory subunit 12/COP9 signalosome complex subunit 4 / Cullin protein, neddylation domain / Cullin protein neddylation domain / PCI/PINT associated module / Cullin repeat-like-containing domain superfamily / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / PCI domain / Proteasome component (PCI) domain / PCI domain profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / Ubiquitin conserved site / Ubiquitin domain signature. / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Tetratricopeptide-like helical domain superfamily / Ubiquitin-like domain superfamily / Zinc finger, RING/FYVE/PHD-type / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Cyclin-dependent kinases regulatory subunit 1 / COP9 signalosome complex subunit 2 / E3 ubiquitin-protein ligase RBX1 / S-phase kinase-associated protein 1 / COP9 signalosome complex subunit 1 / S-phase kinase-associated protein 2 / Cullin-1 / NEDD8 / COP9 signalosome complex subunit 6 / COP9 signalosome complex subunit 5 ...Cyclin-dependent kinases regulatory subunit 1 / COP9 signalosome complex subunit 2 / E3 ubiquitin-protein ligase RBX1 / S-phase kinase-associated protein 1 / COP9 signalosome complex subunit 1 / S-phase kinase-associated protein 2 / Cullin-1 / NEDD8 / COP9 signalosome complex subunit 6 / COP9 signalosome complex subunit 5 / COP9 signalosome complex subunit 8 / COP9 signalosome complex subunit 4 / COP9 signalosome complex subunit 7b / COP9 signalosome complex subunit 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsMosadeghi 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
CitationJournal: 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
DepositionMar 29, 2016-
Header (metadata) releaseApr 6, 2016-
Map releaseApr 6, 2016-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3401.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of CSN5H138A-N8_SCF/Skp2
Voxel sizeX=Y=Z: 1.74 Å
Density
Contour LevelBy AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.08129014 - 0.22193521
Average (Standard dev.)0.00045421 (±0.01186982)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 382.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.741.741.74
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z382.800382.800382.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0810.2220.000

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Supplemental data

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Sample components

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Entire : CSN5H138A-N8_SCF/Skp2

EntireName: 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: Skp1S-phase kinase-associated protein 1
  • Protein or peptide: Skp2
  • Protein or peptide: Cks1

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Supramolecule #1000: CSN5H138A-N8_SCF/Skp2

SupramoleculeName: CSN5H138A-N8_SCF/Skp2 / type: sample / ID: 1000 / Oligomeric state: one CSN monomer and one SCF monomer / Number unique components: 14
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Csn1

MacromoleculeName: 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 weightTheoretical: 55 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 1

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Macromolecule #2: Csn2

MacromoleculeName: 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 weightTheoretical: 51 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 2

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Macromolecule #3: Csn3

MacromoleculeName: 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 weightTheoretical: 47 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 3

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Macromolecule #4: Csn4

MacromoleculeName: 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 weightTheoretical: 46 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 4

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Macromolecule #5: Csn5

MacromoleculeName: 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 weightTheoretical: 37 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 5

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Macromolecule #6: Csn6

MacromoleculeName: 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 weightTheoretical: 36 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 6

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Macromolecule #7: Csn7b

MacromoleculeName: 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 weightTheoretical: 29.6 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 7b

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Macromolecule #8: Csn8

MacromoleculeName: 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 weightTheoretical: 23 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: COP9 signalosome complex subunit 8

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Macromolecule #9: Cul1

MacromoleculeName: 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 weightTheoretical: 89 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: Cullin-1

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Macromolecule #10: Rbx1

MacromoleculeName: 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 weightTheoretical: 12 KDa
Recombinant expressionOrganism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
SequenceUniProtKB: E3 ubiquitin-protein ligase RBX1

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Macromolecule #11: Nedd8

MacromoleculeName: 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 weightTheoretical: 9 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
SequenceUniProtKB: NEDD8

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Macromolecule #12: Skp1

MacromoleculeName: 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 expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
SequenceUniProtKB: S-phase kinase-associated protein 1

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Macromolecule #13: Skp2

MacromoleculeName: 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 expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
SequenceUniProtKB: S-phase kinase-associated protein 2

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Macromolecule #14: Cks1

MacromoleculeName: 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 weightTheoretical: 9.6 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
SequenceUniProtKB: Cyclin-dependent kinases regulatory subunit 1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.6 / Details: 15 mM HEPES, pH 7.6, 100 mM NaCl, 0.5 mM DTT
GridDetails: 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
VitrificationCryogen 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 90 K / Max: 110 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected manually at a nominal magnification of 100,000x
DateMay 12, 2015
Image recordingCategory: 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

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Image processing

CTF correctionDetails: micrographs
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: OTHER / Software - Name: relion, CTFFIND3 / Number images used: 75000
DetailsCTF-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.

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Atomic model buiding 1

Initial modelPDB 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
SoftwareName: Chmiera
Detailssome domains were fitted separately to account for conformational rearrangements, see publication for details
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap

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Atomic model buiding 2

Initial modelPDB 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
SoftwareName: Chmiera
DetailsCsn7b 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
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap

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Atomic model buiding 3

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chmiera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap

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Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

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Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

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