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- PDB-8pql: K48-linked ubiquitin chain formation with a cullin-RING E3 ligase... -

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Entry
Database: PDB / ID: 8pql
TitleK48-linked ubiquitin chain formation with a cullin-RING E3 ligase and Cdc34: NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2-donor UB-acceptor UB-SIL1 peptide
Components
  • Cullin-2
  • E3 ubiquitin-protein ligase RBX1
  • Elongin-B
  • Elongin-C
  • Polyubiquitin-C,Nucleotide exchange factor SIL1
  • Protein fem-1 homolog C
  • Ubiquitin
  • Ubiquitin-conjugating enzyme E2 R2
KeywordsLIGASE / CUL2 / FEM1C / ELOBC / SIL1 / Ubiquitin / Ubiquitin Ligase / Ubiquitin chain formation / Poliubiquitylation
Function / homology
Function and homology information


adenyl-nucleotide exchange factor activity / cotranslational protein targeting to membrane / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / VCB complex ...adenyl-nucleotide exchange factor activity / cotranslational protein targeting to membrane / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / VCB complex / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / elongin complex / positive regulation of protein autoubiquitination / protein neddylation / NEDD8 ligase activity / negative regulation of response to oxidative stress / Cul5-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / ubiquitin-ubiquitin ligase activity / negative regulation of type I interferon production / E2 ubiquitin-conjugating enzyme / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of mitophagy / Prolactin receptor signaling / ubiquitin conjugating enzyme activity / cullin family protein binding / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / protein monoubiquitination / ubiquitin ligase complex / Tat-mediated elongation of the HIV-1 transcript / ubiquitin-like ligase-substrate adaptor activity / Formation of HIV-1 elongation complex containing HIV-1 Tat / Formation of HIV elongation complex in the absence of HIV Tat / protein K48-linked ubiquitination / RNA Polymerase II Transcription Elongation / Nuclear events stimulated by ALK signaling in cancer / Formation of RNA Pol II elongation complex / Maturation of protein E / Maturation of protein E / RNA Polymerase II Pre-transcription Events / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / regulation of cellular response to insulin stimulus / Prevention of phagosomal-lysosomal fusion / positive regulation of TORC1 signaling / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Endosomal Sorting Complex Required For Transport (ESCRT) / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Constitutive Signaling by NOTCH1 HD Domain Mutants / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / negative regulation of insulin receptor signaling pathway / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Regulation of FZD by ubiquitination / Downregulation of ERBB4 signaling / post-translational protein modification / p75NTR recruits signalling complexes / APC-Cdc20 mediated degradation of Nek2A / intrinsic apoptotic signaling pathway / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / Regulation of innate immune responses to cytosolic DNA / NF-kB is activated and signals survival / Downregulation of ERBB2:ERBB3 signaling / Pexophagy / NRIF signals cell death from the nucleus / Regulation of PTEN localization / VLDLR internalisation and degradation / Activated NOTCH1 Transmits Signal to the Nucleus / T cell activation / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Translesion synthesis by REV1 / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Translesion synthesis by POLK / InlB-mediated entry of Listeria monocytogenes into host cell / Downregulation of TGF-beta receptor signaling
Similarity search - Function
Nucleotide exchange factor Fes1 / : / Nucleotide exchange factor Fes1 / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / Cullin protein neddylation domain / : / Cullin, conserved site / Elongin-C ...Nucleotide exchange factor Fes1 / : / Nucleotide exchange factor Fes1 / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / Cullin protein neddylation domain / : / Cullin, conserved site / Elongin-C / Cullin family signature. / Elongin B / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Ankyrin repeat / SKP1/BTB/POZ domain superfamily / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Armadillo-like helical / Ubiquitin family / Ubiquitin homologues / Tetratricopeptide-like helical domain superfamily / Ubiquitin domain profile. / Ubiquitin-like domain / Armadillo-type fold / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
5-azanylpentan-2-one / Polyubiquitin-C / E3 ubiquitin-protein ligase RBX1 / Cullin-2 / Elongin-C / Elongin-B / Ubiquitin-conjugating enzyme E2 R2 / Protein fem-1 homolog C / Nucleotide exchange factor SIL1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsLiwocha, J. / Prabu, J.R. / Kleiger, G. / Schulman, B.A.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases.
Authors: Joanna Liwocha / Jerry Li / Nicholas Purser / Chutima Rattanasopa / Samuel Maiwald / David T Krist / Daniel C Scott / Barbara Steigenberger / J Rajan Prabu / Brenda A Schulman / Gary Kleiger /
Abstract: E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked ...E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.
History
DepositionJul 11, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 14, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 28, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Apr 17, 2024Group: Derived calculations / Structure summary
Category: em_entity_assembly / em_entity_assembly_molwt ...em_entity_assembly / em_entity_assembly_molwt / struct_sheet / struct_sheet_range
Item: _em_entity_assembly.entity_id_list / _em_entity_assembly_molwt.units
Revision 1.4Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Ubiquitin-conjugating enzyme E2 R2
E: Polyubiquitin-C,Nucleotide exchange factor SIL1
H: Protein fem-1 homolog C
K: E3 ubiquitin-protein ligase RBX1
U: Ubiquitin
A: Cullin-2
D: Elongin-C
G: Elongin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)377,62512
Polymers377,3278
Non-polymers2974
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15260 Å2
ΔGint-91 kcal/mol
Surface area92390 Å2
MethodPISA

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Components

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Protein , 8 types, 8 molecules CEHKUADG

#1: Protein Ubiquitin-conjugating enzyme E2 R2 / E2 ubiquitin-conjugating enzyme R2 / Ubiquitin carrier protein R2 / Ubiquitin-conjugating enzyme E2- ...E2 ubiquitin-conjugating enzyme R2 / Ubiquitin carrier protein R2 / Ubiquitin-conjugating enzyme E2-CDC34B / Ubiquitin-protein ligase R2


Mass: 27190.932 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2R2, CDC34B, UBC3B / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: Q712K3, E2 ubiquitin-conjugating enzyme
#2: Protein Polyubiquitin-C,Nucleotide exchange factor SIL1 / BiP-associated protein / BAP


Mass: 79226.711 Da / Num. of mol.: 1 / Mutation: K48C,S455P L456T L457Q K458G E459R L460A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC, SIL1, UNQ545/PRO836 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0CG48, UniProt: Q9H173
#3: Protein Protein fem-1 homolog C / FEM1c / FEM1-gamma


Mass: 68767.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FEM1C, KIAA1785 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q96JP0
#4: Protein E3 ubiquitin-protein ligase RBX1 / E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / ...E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / Rbx1 / Regulator of cullins 1 / ROC1


Mass: 12289.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBX1, RNF75, ROC1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P62877, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase
#5: Protein Ubiquitin


Mass: 77120.398 Da / Num. of mol.: 1 / Mutation: K48C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0CG48
#6: Protein Cullin-2 / CUL-2


Mass: 87098.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q13617
#7: Protein Elongin-C / EloC / Elongin 15 kDa subunit / RNA polymerase II transcription factor SIII subunit C / SIII p15 / ...EloC / Elongin 15 kDa subunit / RNA polymerase II transcription factor SIII subunit C / SIII p15 / Transcription elongation factor B polypeptide 1


Mass: 12485.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOC, TCEB1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q15369
#8: Protein Elongin-B / EloB / Elongin 18 kDa subunit / RNA polymerase II transcription factor SIII subunit B / SIII p18 / ...EloB / Elongin 18 kDa subunit / RNA polymerase II transcription factor SIII subunit B / SIII p18 / Transcription elongation factor B polypeptide 2


Mass: 13147.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOB, TCEB2 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q15370

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Non-polymers , 2 types, 4 molecules

#9: Chemical ChemComp-SY8 / 5-azanylpentan-2-one


Mass: 101.147 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H11NO
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1K48-linked ubiquitin chain formation with a cullin-RING E3 ligase and CDC34:NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2-donor UB-acceptor UB-SIL1 peptideCOMPLEX#1-#80MULTIPLE SOURCES
2RING E3 ligase (RBX1) and Cullin-2 (CUL2)COMPLEX#4, #61RECOMBINANT
3CDC34, NEDD8, ELOB, ELOC, FEM1C with UBE2R2-donor UB-acceptor UB-SIL1 peptide and K48-linked ubiquitin chainCOMPLEX#1-#3, #5, #7-#81RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
33Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61956 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413829
ELECTRON MICROSCOPYf_angle_d0.71918849
ELECTRON MICROSCOPYf_dihedral_angle_d4.5181949
ELECTRON MICROSCOPYf_chiral_restr0.0422224
ELECTRON MICROSCOPYf_plane_restr0.0062418

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