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- PDB-9b5i: Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic... -

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Basic information

Entry
Database: PDB / ID: 9b5i
TitleUbiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - cluster 4 map and model (Ub(A)-AMP/PPi/Mg)
Components
  • Polyubiquitin
  • Ubiquitin
  • Ubiquitin-activating enzyme E1 1
  • Ubiquitin-conjugating enzyme E2 4
KeywordsLIGASE / UBIQUITIN / E1 / E2 / UBA1 / UBC4 / TRANSTHIOESTERIFICATION / THIOESTER / TRANSTHIOLATION / TETRAHEDRAL INTERMEDIATE / ADENYLATION / INHIBITOR / NUCLEUS / PHOSPHOPROTEIN / UBL CONJUGATION PATHWAY / UBL / ATP ATP-BINDING / AMP / NUCLEOTIDE-BINDING / ISOPEPTIDE BOND
Function / homology
Function and homology information


PINK1-PRKN Mediated Mitophagy / Hedgehog ligand biogenesis / Regulation of necroptotic cell death / Josephin domain DUBs / ER Quality Control Compartment (ERQC) / Interleukin-1 signaling / Endosomal Sorting Complex Required For Transport (ESCRT) / RAS processing / Pexophagy / Regulation of pyruvate metabolism ...PINK1-PRKN Mediated Mitophagy / Hedgehog ligand biogenesis / Regulation of necroptotic cell death / Josephin domain DUBs / ER Quality Control Compartment (ERQC) / Interleukin-1 signaling / Endosomal Sorting Complex Required For Transport (ESCRT) / RAS processing / Pexophagy / Regulation of pyruvate metabolism / Translesion synthesis by REV1 / Translesion Synthesis by POLH / Translesion synthesis by POLK / Translesion synthesis by POLI / MAPK6/MAPK4 signaling / UCH proteinases / Orc1 removal from chromatin / CDK-mediated phosphorylation and removal of Cdc6 / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Ubiquitin-dependent degradation of Cyclin D / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / KEAP1-NFE2L2 pathway / Formation of Incision Complex in GG-NER / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Gap-filling DNA repair synthesis and ligation in GG-NER / Aggrephagy / Ub-specific processing proteases / Dual Incision in GG-NER / Neddylation / Metalloprotease DUBs / Regulation of PTEN localization / Regulation of PTEN stability and activity / nuclear-transcribed mRNA catabolic process, meiosis-specific transcripts / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / L13a-mediated translational silencing of Ceruloplasmin expression / Formation of a pool of free 40S subunits / GTP hydrolysis and joining of the 60S ribosomal subunit / SRP-dependent cotranslational protein targeting to membrane / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Peroxisomal protein import / E3 ubiquitin ligases ubiquitinate target proteins / nuclear SCF ubiquitin ligase complex / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Antigen processing: Ubiquitination & Proteasome degradation / E1 ubiquitin-activating enzyme / ubiquitin activating enzyme activity / SREBP signaling pathway / positive regulation of mitotic metaphase/anaphase transition / enzyme inhibitor activity / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / E2 ubiquitin-conjugating enzyme / ubiquitin conjugating enzyme activity / ubiquitin ligase complex / meiotic cell cycle / modification-dependent protein catabolic process / protein polyubiquitination / ubiquitin-protein transferase activity / protein tag activity / ribosome biogenesis / ubiquitin-dependent protein catabolic process / cytoplasmic translation / proteasome-mediated ubiquitin-dependent protein catabolic process / cytosolic large ribosomal subunit / protein ubiquitination / structural constituent of ribosome / DNA damage response / ubiquitin protein ligase binding / nucleolus / magnesium ion binding / ATP hydrolysis activity / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ubiquitin-activating enzyme E1 / Ubiquitin-activating enzyme E1, C-terminal / Ubiquitin-activating enzyme E1, FCCH domain / Ubiquitin-activating enzyme E1, four-helix bundle / Ubiquitin-activating enzyme E1, C-terminal domain superfamily / Ubiquitin-activating enzyme E1, SCCH domain / Ubiquitin-activating enzyme E1, FCCH domain superfamily / Ubiquitin fold domain / Ubiquitin-activating enzyme E1 FCCH domain / Ubiquitin-activating enzyme E1 four-helix bundle ...Ubiquitin-activating enzyme E1 / Ubiquitin-activating enzyme E1, C-terminal / Ubiquitin-activating enzyme E1, FCCH domain / Ubiquitin-activating enzyme E1, four-helix bundle / Ubiquitin-activating enzyme E1, C-terminal domain superfamily / Ubiquitin-activating enzyme E1, SCCH domain / Ubiquitin-activating enzyme E1, FCCH domain superfamily / Ubiquitin fold domain / Ubiquitin-activating enzyme E1 FCCH domain / Ubiquitin-activating enzyme E1 four-helix bundle / Ubiquitin-activating enzyme e1 C-terminal domain / Ubiquitin-activating enzyme, SCCH domain / Ubiquitin-activating enzyme, SCCH domain / Ubiquitin/SUMO-activating enzyme E1-like / Ubiquitin-activating enzyme E1, inactive adenylation domain, subdomain 1 / Ubiquitin-activating enzyme E1, Cys active site / Ubiquitin-activating enzyme active site. / ThiF/MoeB/HesA family / THIF-type NAD/FAD binding fold / ThiF family / Ubiquitin-activating enzyme / 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 / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
: / ADENOSINE MONOPHOSPHATE / PYROPHOSPHATE 2- / Ubiquitin-activating enzyme E1 1 / Polyubiquitin / Ubiquitin-ribosomal protein eL40B fusion protein / Ubiquitin-conjugating enzyme E2 4
Similarity search - Component
Biological speciesSchizosaccharomyces pombe 972h- (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKochanczyk, T. / Lima, C.D.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118080 United States
CitationJournal: Nature / Year: 2024
Title: Structural basis for transthiolation intermediates in the ubiquitin pathway.
Authors: Tomasz Kochańczyk / Zachary S Hann / Michaelyn C Lux / Avelyn Mae V Delos Reyes / Cheng Ji / Derek S Tan / Christopher D Lima /
Abstract: Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin ...Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters. How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1-Ub(T)-E2 and E2-Ub(T)-E3 complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.
History
DepositionMar 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Sep 18, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ubiquitin-activating enzyme E1 1
B: Polyubiquitin
C: Ubiquitin-conjugating enzyme E2 4
D: Ubiquitin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,8029
Polymers146,1464
Non-polymers6565
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 4 molecules ABCD

#1: Protein Ubiquitin-activating enzyme E1 1 / Poly(A)+ RNA transport protein 3


Mass: 111764.047 Da / Num. of mol.: 1 / Fragment: residues 13-1012
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe 972h- (yeast)
Strain: 972 / ATCC 24843 / Gene: ptr3, SPBC1604.21c, SPBC211.09 / Plasmid: pSMT3 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O94609, E1 ubiquitin-activating enzyme
#2: Protein Polyubiquitin


Mass: 8568.769 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe 972h- (yeast)
Strain: 972 / ATCC 24843 / Gene: ubi4, SPBC337.08c / Plasmid: pET3C / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0CG72
#3: Protein Ubiquitin-conjugating enzyme E2 4 / E2 ubiquitin-conjugating enzyme 4 / Ubiquitin carrier protein 4 / Ubiquitin-protein ligase 4


Mass: 17043.336 Da / Num. of mol.: 1 / Mutation: C21S,C107S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe 972h- (yeast)
Strain: 972 / ATCC 24843 / Gene: ubc4, SPBC119.02 / Plasmid: pET29b+ / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P46595, E2 ubiquitin-conjugating enzyme
#4: Protein Ubiquitin


Mass: 8769.948 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe 972h- (yeast)
Strain: 972 / ATCC 24843 / Gene: uep1, ubi2, SPAC1805.12c / Plasmid: pTXB1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0CH07

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Non-polymers , 4 types, 5 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-POP / PYROPHOSPHATE 2-


Mass: 175.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H2O7P2
#7: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Comment: AMP*YM
#8: Chemical ChemComp-A1AIV / 4-aminobutanenitrile


Mass: 84.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H8N2 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

ComponentName: Covalent E1-Ub-E2 transthiolation intermediate mimic complex with second Ub bound to E1 (doubly Ub-loaded E1-Ub-E2 complex)
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Schizosaccharomyces pombe 972h- (yeast) / Strain: 972 / ATCC 24843
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.2
Details: 20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, 1 mM ATP, 0.05% CHAPSO
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris HCl bufferC4H12NO31
2100 mMSodium chlorideNaCl1
35 mMMagnesium chlorideMgCl21
41 mMAdenosine triphosphateC10H16N5O13P31
50.5 mg/mlCHAPSOC32H59N2O8S1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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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: 2500 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2PHENIX1.20.1_4487:model refinement
3SerialEMimage acquisition
4cryoSPARCmasking
5GctfCTF correction
6PHENIXmodel fitting
7RELIONother
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324446 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039745
ELECTRON MICROSCOPYf_angle_d0.44313195
ELECTRON MICROSCOPYf_dihedral_angle_d9.3863639
ELECTRON MICROSCOPYf_chiral_restr0.041471
ELECTRON MICROSCOPYf_plane_restr0.0031709

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