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- PDB-9b5p: Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic... -

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Basic information

Entry
Database: PDB / ID: 9b5p
TitleUbiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - cluster 1 map and model (ATP/Mg)
Components
  • 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


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 / E1 ubiquitin-activating enzyme / ubiquitin activating enzyme activity / SREBP signaling pathway / positive regulation of mitotic metaphase/anaphase transition / E2 ubiquitin-conjugating enzyme ...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 / E1 ubiquitin-activating enzyme / ubiquitin activating enzyme activity / SREBP signaling pathway / positive regulation of mitotic metaphase/anaphase transition / E2 ubiquitin-conjugating enzyme / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / ubiquitin conjugating enzyme activity / protein K48-linked ubiquitination / ubiquitin ligase complex / modification-dependent protein catabolic process / protein polyubiquitination / ubiquitin-protein transferase activity / protein tag activity / ribosome biogenesis / ubiquitin-dependent protein catabolic process / cytosolic large ribosomal subunit / cytoplasmic translation / protein ubiquitination / structural constituent of ribosome / ubiquitin protein ligase binding / DNA damage response / nucleolus / magnesium ion binding / ATP hydrolysis activity / ATP binding / nucleus / cytoplasm / cytosol
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 / Ubiquitin-activating enzyme / THIF-type NAD/FAD binding fold / ThiF family / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / 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-5'-TRIPHOSPHATE / Ubiquitin-activating enzyme E1 1 / 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: 3.08 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-activating enzyme E1 1
C: Ubiquitin-conjugating enzyme E2 4
D: Ubiquitin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)138,2177
Polymers137,5773
Non-polymers6404
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 , 3 types, 3 molecules ACD

#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 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
#3: 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 , 3 types, 4 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: 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 (singly 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: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30181 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029140
ELECTRON MICROSCOPYf_angle_d0.44312390
ELECTRON MICROSCOPYf_dihedral_angle_d8.6653393
ELECTRON MICROSCOPYf_chiral_restr0.0411374
ELECTRON MICROSCOPYf_plane_restr0.0031604

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