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- EMDB-17127: Cryo-EM structure of human tRNA ligase RTCB in complex with human... -

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Basic information

Entry
Database: EMDB / ID: EMD-17127
TitleCryo-EM structure of human tRNA ligase RTCB in complex with human PYROXD1.
Map dataUnsharpened map
Sample
  • Complex: Cryo-EM structure of human RTCB in complex with human PYROXD1.
    • Protein or peptide: RNA-splicing ligase RtcB homolog
    • Protein or peptide: Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1
  • Ligand: MAGNESIUM ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: DIHYDROFLAVINE-ADENINE DINUCLEOTIDE
KeywordstRNA ligase / PYROXD1 / RTCB / Flavoprotein / Ligase
Function / homology
Function and homology information


tRNA-splicing ligase complex / Oxidoreductases; Acting on a sulfur group of donors; With NAD+ or NADP+ as acceptor / 3'-phosphate/5'-hydroxy nucleic acid ligase / RNA ligase (GTP) activity / tRNA splicing, via endonucleolytic cleavage and ligation / vinculin binding / tRNA processing in the nucleus / sarcomere / placenta development / nuclear envelope ...tRNA-splicing ligase complex / Oxidoreductases; Acting on a sulfur group of donors; With NAD+ or NADP+ as acceptor / 3'-phosphate/5'-hydroxy nucleic acid ligase / RNA ligase (GTP) activity / tRNA splicing, via endonucleolytic cleavage and ligation / vinculin binding / tRNA processing in the nucleus / sarcomere / placenta development / nuclear envelope / cellular response to oxidative stress / in utero embryonic development / oxidoreductase activity / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / GTP binding / RNA binding / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
NADH-rubredoxin oxidoreductase, C-terminal / Rubredoxin NAD+ reductase C-terminal domain / RNA-splicing ligase RtcB homologue, eukaryotic / Uncharacterized protein family UPF0027 signature. / RNA-splicing ligase, RtcB / tRNA-splicing ligase RtcB-like superfamily / tRNA-splicing ligase RtcB / : / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain ...NADH-rubredoxin oxidoreductase, C-terminal / Rubredoxin NAD+ reductase C-terminal domain / RNA-splicing ligase RtcB homologue, eukaryotic / Uncharacterized protein family UPF0027 signature. / RNA-splicing ligase, RtcB / tRNA-splicing ligase RtcB-like superfamily / tRNA-splicing ligase RtcB / : / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1 / RNA-splicing ligase RtcB homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLoeff L / Kroupova A / Asanovic I / Boneberg F / Pfleiderer MM / Ferdigg A / Ackle F / Martinez J / Jinek M
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567 Switzerland
Citation
Journal: Nat Struct Mol Biol / Year: 2025
Title: Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation.
Authors: Luuk Loeff / Alena Kroupova / Igor Asanović / Franziska M Boneberg / Moritz M Pfleiderer / Luca Riermeier / Alexander Leitner / Andrè Ferdigg / Fabian Ackle / Javier Martinez / Martin Jinek /
Abstract: The metazoan tRNA ligase complex (tRNA-LC) has essential roles in tRNA biogenesis and unfolded protein response. Its catalytic subunit RTCB contains a conserved active-site cysteine that is ...The metazoan tRNA ligase complex (tRNA-LC) has essential roles in tRNA biogenesis and unfolded protein response. Its catalytic subunit RTCB contains a conserved active-site cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of human tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here, we report a cryogenic electron microscopic structure of the human RTCB-PYROXD1 complex, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its carboxy-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment, while reoxidation of PYROXD1 enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of the tRNA-LC under aerobic conditions.
#1: Journal: BioRxiv / Year: 2023
Title: Mechanistic basis for oxidative stress protection of the human tRNA ligase complex by the oxidoreductase PYROXD1
Authors: Loeff L / Kroupova A / Asanovic I / Boneberg F / Pfleiderer MM / Ferdigg A / Ackle F / Martinez J / Jinek M
History
DepositionApr 14, 2023-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17127.png

Downloads & links

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Map

FileDownload / File: emd_17127.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 288 pix.
= 187.2 Å		0.65 Å/pix.
x 288 pix.
= 187.2 Å		0.65 Å/pix.
x 288 pix.
= 187.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0289
Minimum - Maximum-0.08911497 - 0.17467375
Average (Standard dev.)-0.0001379971 (±0.0072747846)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 187.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17127_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_17127_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened map

Fileemd_17127_half_map_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened map

Fileemd_17127_half_map_2.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of human RTCB in complex with human PYROXD1.

EntireName: Cryo-EM structure of human RTCB in complex with human PYROXD1.
Components
  • Complex: Cryo-EM structure of human RTCB in complex with human PYROXD1.
    • Protein or peptide: RNA-splicing ligase RtcB homolog
    • Protein or peptide: Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1
  • Ligand: MAGNESIUM ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: DIHYDROFLAVINE-ADENINE DINUCLEOTIDE

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Supramolecule #1: Cryo-EM structure of human RTCB in complex with human PYROXD1.

SupramoleculeName: Cryo-EM structure of human RTCB in complex with human PYROXD1.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: In vitro reconstituted protein complex
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: RNA-splicing ligase RtcB homolog

MacromoleculeName: RNA-splicing ligase RtcB homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: 3'-phosphate/5'-hydroxy nucleic acid ligase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.556445 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SNAMSRSYND ELQFLEKINK NCWRIKKGFV PNMQVEGVFY VNDALEKLMF EELRNACRGG GVGGFLPAMK QIGNVAALPG IVHRSIGLP DVHSGYGFAI GNMAAFDMND PEAVVSPGGV GFDINCGVRL LRTNLDESDV QPVKEQLAQA MFDHIPVGVG S KGVIPMNA ...String:
SNAMSRSYND ELQFLEKINK NCWRIKKGFV PNMQVEGVFY VNDALEKLMF EELRNACRGG GVGGFLPAMK QIGNVAALPG IVHRSIGLP DVHSGYGFAI GNMAAFDMND PEAVVSPGGV GFDINCGVRL LRTNLDESDV QPVKEQLAQA MFDHIPVGVG S KGVIPMNA KDLEEALEMG VDWSLREGYA WAEDKEHCEE YGRMLQADPN KVSARAKKRG LPQLGTLGAG NHYAEIQVVD EI FNEYAAK KMGIDHKGQV CVMIHSGSRG LGHQVATDAL VAMEKAMKRD KIIVNDRQLA CARIASPEGQ DYLKGMAAAG NYA WVNRSS MTFLTRQAFA KVFNTTPDDL DLHVIYDVSH NIAKVEQHVV DGKERTLLVH RKGSTRAFPP HHPLIAVDYQ LTGQ PVLIG GTMGTCSYVL TGTEQGMTET FGTTCHGAGR ALSRAKSRRN LDFQDVLDKL ADMGIAIRVA SPKLVMEEAP ESYKN VTDV VNTCHDAGIS KKAIKLRPIA VIKG

UniProtKB: RNA-splicing ligase RtcB homolog

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Macromolecule #2: Pyridine nucleotide-disulfide oxidoreductase domain-containing pr...

MacromoleculeName: Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Oxidoreductases; Acting on a sulfur group of donors; With NAD+ or NADP+ as acceptor
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 56.016859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPMEAARPPP TAGKFVVVGG GIAGVTCAEQ LATHFPSEDI LLVTASPVIK AVTNFKQISK ILEEFDVEEQ SSTMLGKRFP NIKVIESGV KQLKSEEHCI VTEDGNQHVY KKLCLCAGAK PKLICEGNPY VLGIRDTDSA QEFQKQLTKA KRIMIIGNGG I ALELVYEI ...String:
GPMEAARPPP TAGKFVVVGG GIAGVTCAEQ LATHFPSEDI LLVTASPVIK AVTNFKQISK ILEEFDVEEQ SSTMLGKRFP NIKVIESGV KQLKSEEHCI VTEDGNQHVY KKLCLCAGAK PKLICEGNPY VLGIRDTDSA QEFQKQLTKA KRIMIIGNGG I ALELVYEI EGCEVIWAIK DKAIGNTFFD AGAAEFLTSK LIAEKSEAKI AHKRTRYTTE GRKKEARSKS KADNVGSALG PD WHEGLNL KGTKEFSHKI HLETMCEVKK IYLQDEFRIL KKKSFTFPRD HKSVTADTEM WPVYVELTNE KIYGCDFIVS ATG VTPNVE PFLHGNSFDL GEDGGLKVDD HMHTSLPDIY AAGDICTTSW QLSPVWQQMR LWTQARQMGW YAAKCMAAAS SGDS IDMDF SFELFAHVTK FFNYKVVLLG KYNAQGLGSD HELMLRCTKG REYIKVVMQN GRMMGAVLIG ETDLEETFEN LILNQ MNLS SYGEDLLDPN IDIEDYFD

UniProtKB: Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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Macromolecule #5: DIHYDROFLAVINE-ADENINE DINUCLEOTIDE

MacromoleculeName: DIHYDROFLAVINE-ADENINE DINUCLEOTIDE / type: ligand / ID: 5 / Number of copies: 1 / Formula: FDA
Molecular weightTheoretical: 787.566 Da
Chemical component information

ChemComp-FDA:
DIHYDROFLAVINE-ADENINE DINUCLEOTIDE

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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

Concentration0.6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
5.0 mMMgCl2Magnesium chloride
0.5 mMC9H15O6PTCEP
0.5 mMC21H27N7O14P2NADH

Details: Dataset 1: 20 mM HEPES pH 8.0, 150 mM NaCl, 0.5 mM TCEP, 5 mM MgCl2 and 0.5 mM NADH, 0.01% Octyl-beta-Glucoside Dataset 2: 20 mM HEPES pH 8.0, 150 mM NaCl, 0.5 mM TCEP, 5 mM MgCl2 and 0.5 mM NADH
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThe complex was purified over size exclusion chromatography, prior to grid freezing. Protein concentration per dataset: Dataset 1: 1.5 mg/ ml Dataset 2: 0.6 mg/ ml

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 5114 / #0 - Average electron dose: 64.592 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 9302 / #1 - Average electron dose: 66.459 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: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
CTF correctionSoftware - Name: cryoSPARC (ver. 2.3.3) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 288325
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 1
FSC plot (resolution estimation)

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