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- PDB-9j5o: Cryo-EM structure of TrhO from B. subtilis complexed with tRNA Ala -

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Basic information

Entry
Database: PDB / ID: 9j5o
TitleCryo-EM structure of TrhO from B. subtilis complexed with tRNA Ala
Components
  • RNA (76-MER)
  • tRNA uridine(34) hydroxylase
KeywordsRNA BINDING PROTEIN / Hydroxylase / tRNA post-transcriptional modification / TrhO / ho5U / Hydroxylase-RNA complex
Function / homology
Function and homology information


Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen / tRNA modification / oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen
Similarity search - Function
Rhodanase, C-terminal / Rhodanase C-terminal / tRNA uridine(34) hydroxylase / tRNA uridine(34) hydroxylase, N-terminal / UPF0176 acylphosphatase like domain / Rhodanese Homology Domain / Rhodanese-like domain / Rhodanese domain profile. / Rhodanese-like domain superfamily / Rhodanese-like domain
Similarity search - Domain/homology
RNA / RNA (> 10) / tRNA uridine(34) hydroxylase
Similarity search - Component
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsShin, K. / Kim, J.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2021R1A2C2009773 Korea, Republic Of
CitationJournal: Nat Chem Biol / Year: 2026
Title: Unconventional monooxygenation by the O-dependent tRNA wobble uridine hydroxylase TrhO.
Authors: Kiroo Shin / Da Bean Han / Hyun Woo Kim / Jungwook Kim /
Abstract: Modifications at the wobble position of transfer RNA (tRNA) are critical for accurate codon recognition and efficient translation. 5-Hydroxyuridine serves as a key intermediate for more complex ...Modifications at the wobble position of transfer RNA (tRNA) are critical for accurate codon recognition and efficient translation. 5-Hydroxyuridine serves as a key intermediate for more complex wobble uridine derivatives commonly found in bacterial tRNAs and is synthesized by either prephenate-dependent TrhP or dioxygen-dependent TrhO. Despite its biological importance, structural and mechanistic insights into these enzymes have remained elusive. Here, we report the cryo-electron microscopy structure of Bacillus subtilis TrhO-tRNA complex. Combined with biochemical analyses, our results reveal that TrhO functions without any metal or organic cofactor, unlike most other oxygenases. We propose that the conserved C179 reacts with dioxygen to form a thiohydroperoxy intermediate, which is cleaved to produce 5-hydroxyuridine and a sulfenic acid at C179. The oxidized cysteine subsequently forms a disulfide bond with the adjacent C185, protecting the catalytic cysteine from irreversible overoxidation. These findings broaden our understanding of cofactor-independent dioxygen use in aromatic ring hydroxylation.
History
DepositionAug 13, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 21, 2026Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Jan 21, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 4, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.1Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin / Data content type: EM metadata / EM metadata / EM metadata
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: tRNA uridine(34) hydroxylase
B: RNA (76-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,0943
Polymers65,0282
Non-polymers651
Water00
1


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

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Components

#1: Protein tRNA uridine(34) hydroxylase / tRNA hydroxylation protein O


Mass: 40520.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Gene: trhO, ybfQ, BSU02330 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: O31457, Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen
#2: RNA chain RNA (76-MER)


Mass: 24507.547 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestY
Has protein modificationN

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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: Bacillus subtilis TrhO complexed with Alanine-specific tRNA
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.065 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 25 mM HEPES (pH 7.5), 150 mM NaCl, 0.2 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
30.2 mMTCEPC9H15O6P1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 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 magnification: 130000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 7.15 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118262 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034332
ELECTRON MICROSCOPYf_angle_d0.4856197
ELECTRON MICROSCOPYf_dihedral_angle_d22.5321436
ELECTRON MICROSCOPYf_chiral_restr0.035726
ELECTRON MICROSCOPYf_plane_restr0.004541

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