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- PDB-9b5b: Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate ... -

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Basic information

Entry
Database: PDB / ID: 9b5b
TitleUbiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 7
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 / E3 ubiquitin ligases ubiquitinate target proteins / Peroxisomal protein import / 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 / E3 ubiquitin ligases ubiquitinate target proteins / Peroxisomal protein import / 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 / E2 ubiquitin-conjugating enzyme / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / cell division site / ubiquitin conjugating enzyme activity / protein K48-linked ubiquitination / ubiquitin ligase complex / modification-dependent protein catabolic process / protein polyubiquitination / protein tag activity / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / ribosome biogenesis / ubiquitin-dependent protein catabolic process / cytosolic large ribosomal subunit / cytoplasmic translation / 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 / WW/rsp5/WWP domain signature. ...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 / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW domain / 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: 3.31 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: To be published
Title: Structural basis for transthiolation intermediates in the ubiquitin pathway
Authors: Kochanczyk, T. / Hann, Z.S. / Lux, M.C. / Reyes, A.M.V.D. / Ji, C. / Tan, D.S. / Lima, C.D.
History
DepositionMar 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94260 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034983
ELECTRON MICROSCOPYf_angle_d0.4346747
ELECTRON MICROSCOPYf_dihedral_angle_d9.2521877
ELECTRON MICROSCOPYf_chiral_restr0.039737
ELECTRON MICROSCOPYf_plane_restr0.003873

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