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- EMDB-61148: Cryo-EM structure of TrhO from B. subtilis complexed with tRNA Ala -

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Basic information

Entry
Database: EMDB / ID: EMD-61148
TitleCryo-EM structure of TrhO from B. subtilis complexed with tRNA Ala
Map dataDeepEMhancer sharpened map
Sample
  • Complex: Bacillus subtilis TrhO complexed with Alanine-specific tRNA
    • Protein or peptide: tRNA uridine(34) hydroxylase
    • RNA: RNA (76-MER)
  • Ligand: ZINC ION
KeywordsHydroxylase / tRNA post-transcriptional modification / TrhO / ho5U / Hydroxylase-RNA complex / RNA BINDING PROTEIN
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
tRNA uridine(34) hydroxylase
Similarity search - Component
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsShin K / Kim J
Funding support Korea, Republic Of, 1 items
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, 2024-
Header (metadata) releaseJan 21, 2026-
Map releaseJan 21, 2026-
UpdateFeb 4, 2026-
Current statusFeb 4, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61148.png

Downloads & links

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Map

FileDownload / File: emd_61148.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 360 pix.
= 324.468 Å		0.9 Å/pix.
x 360 pix.
= 324.468 Å		0.9 Å/pix.
x 360 pix.
= 324.468 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9013 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055
Minimum - Maximum-0.0017972518 - 1.6977125
Average (Standard dev.)0.0001833875 (±0.009905112)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 324.46802 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_61148_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_61148_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bacillus subtilis TrhO complexed with Alanine-specific tRNA

EntireName: Bacillus subtilis TrhO complexed with Alanine-specific tRNA
Components
  • Complex: Bacillus subtilis TrhO complexed with Alanine-specific tRNA
    • Protein or peptide: tRNA uridine(34) hydroxylase
    • RNA: RNA (76-MER)
  • Ligand: ZINC ION

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Supramolecule #1: Bacillus subtilis TrhO complexed with Alanine-specific tRNA

SupramoleculeName: Bacillus subtilis TrhO complexed with Alanine-specific tRNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 65 KDa

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Macromolecule #1: tRNA uridine(34) hydroxylase

MacromoleculeName: tRNA uridine(34) hydroxylase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 40.520547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGSHHHHHH GMASMTGGQQ MGRSGDDDDK MEKQYRVLLY YKYVPIEDPE AFREQHLAFC KELGLLGRIL VSSEGINGTV SGTVEQTEK YMETMKADPR FADMVFKIDE AEGHAFKKIF VRHKKELVTL RLEDDVDPNE TTGQHLKPAE FYEKMQDPNT I VIDARNDY ...String:
MAGSHHHHHH GMASMTGGQQ MGRSGDDDDK MEKQYRVLLY YKYVPIEDPE AFREQHLAFC KELGLLGRIL VSSEGINGTV SGTVEQTEK YMETMKADPR FADMVFKIDE AEGHAFKKIF VRHKKELVTL RLEDDVDPNE TTGQHLKPAE FYEKMQDPNT I VIDARNDY EYDLGHFRGA VRPDIEAFRE LPEWIEEHKD MLEGKKILTY CTGGVRCEKF SGWLVKQGFE DVAQLDGGIV TY GKDPEVQ GKLWDGQCYV FDERISVPVN RVEHVIVGKD YFTGEPCERY VNCANPSCNK KMICTPENEY KYMRSCSHEC RTN PRNLYV KEHNMTEEEV NARLAAIETE DHAAAE

UniProtKB: tRNA uridine(34) hydroxylase

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Macromolecule #2: RNA (76-MER)

MacromoleculeName: RNA (76-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 24.507547 KDa
SequenceString:
GGGGCCUUAG CUCAGCUGGG AGAGCGCCUG CUU(A1L3O)GCACGC AGGAGGUCAG CGGUUCGAUC CCGCUAGGCU CCAC CA

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
0.2 mMC9H15O6PTCEP

Details: 25 mM HEPES (pH 7.5), 150 mM NaCl, 0.2 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average exposure time: 7.15 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 118262
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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