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- PDB-9b57: Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate ... -

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Basic information

Entry
Database: PDB / ID: 9b57
TitleUbiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 3
Components
  • E3 ubiquitin-protein ligase pub2
  • Ubiquitin
  • Ubiquitin-conjugating enzyme E2 4
KeywordsLIGASE / UBIQUITIN / E2 / E3 / HECT / NEDD4 / RSP5 / PUB2 / 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


RHOQ GTPase cycle / RHOU GTPase cycle / Regulation of PTEN localization / Regulation of PTEN stability and activity / cytoplasm to vacuole targeting by the NVT pathway / cell cortex of cell tip / Peroxisomal protein import / E3 ubiquitin ligases ubiquitinate target proteins / nuclear SCF ubiquitin ligase complex / Synthesis of active ubiquitin: roles of E1 and E2 enzymes ...RHOQ GTPase cycle / RHOU GTPase cycle / Regulation of PTEN localization / Regulation of PTEN stability and activity / cytoplasm to vacuole targeting by the NVT pathway / cell cortex of cell tip / 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 / SREBP signaling pathway / positive regulation of mitotic metaphase/anaphase transition / HECT-type E3 ubiquitin transferase / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / E2 ubiquitin-conjugating enzyme / cell division site / ubiquitin conjugating enzyme activity / ubiquitin ligase complex / modification-dependent protein catabolic process / protein polyubiquitination / ubiquitin-protein transferase activity / protein tag activity / ubiquitin protein ligase activity / ribosome biogenesis / ubiquitin-dependent protein catabolic process / cytoplasmic translation / cytosolic large ribosomal subunit / protein ubiquitination / structural constituent of ribosome / ubiquitin protein ligase binding / nucleolus / ATP binding / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
E3 ubiquitin-protein ligase, SMURF1 type / : / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / WW domain ...E3 ubiquitin-protein ligase, SMURF1 type / : / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / WW domain / WW/rsp5/WWP domain signature. / C2 domain / C2 domain profile. / WW domain superfamily / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / WW domain / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / C2 domain 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
: / Ubiquitin-ribosomal protein eL40B fusion protein / Ubiquitin-conjugating enzyme E2 4 / E3 ubiquitin-protein ligase pub2
Similarity search - Component
Biological speciesSchizosaccharomyces pombe 972h- (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 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 14, 2024Group: Data collection / Database references / Category: citation / 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.year / _em_admin.last_update
Revision 1.2Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _em_admin.last_update
Revision 1.3Sep 4, 2024Group: Data collection / Database references / Category: citation_author / em_admin
Item: _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.4Sep 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.5Sep 18, 2024Group: Data collection / Category: em_admin / Item: _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-conjugating enzyme E2 4
B: Ubiquitin
C: E3 ubiquitin-protein ligase pub2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)70,1294
Polymers70,0453
Non-polymers841
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: 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
Details: C-terminal GGLVPR is a residual artifact after thrombin cleavage of affinity tag
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
#2: Protein Ubiquitin


Mass: 8769.948 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal GSGG is a residual artifact after TEV protease cleavage of affinity tag
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
#3: Protein E3 ubiquitin-protein ligase pub2 / HECT-type E3 ubiquitin transferase pub2


Mass: 44231.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal SHM is a residual artifact after cleaving the affinity tag
Source: (gene. exp.) Schizosaccharomyces pombe 972h- (yeast)
Strain: 972 / ATCC 24843 / Gene: pub2, SPAC1805.15c / Plasmid: pSMT3 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9UTG2, HECT-type E3 ubiquitin transferase
#4: Chemical ChemComp-A1AIV / 4-aminobutanenitrile


Mass: 84.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H8N2 / Feature type: SUBJECT OF INVESTIGATION
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 E2-Ub-E3 HECT transthiolation intermediate mimic 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, 0.1% CHAPSO
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris HCl bufferC4H12NO31
2100 mMSodium chlorideNaCl1
31 mg/mlCHAPSOC32H59N2O8S1
SpecimenConc.: 4.5 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: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169620 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0024983
ELECTRON MICROSCOPYf_angle_d0.4716747
ELECTRON MICROSCOPYf_dihedral_angle_d10.7181877
ELECTRON MICROSCOPYf_chiral_restr0.039737
ELECTRON MICROSCOPYf_plane_restr0.003873

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