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- PDB-8orj: Cryo-EM structure of human tRNA ligase RTCB in complex with human... -

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Basic information

Entry
Database: PDB / ID: 8orj
TitleCryo-EM structure of human tRNA ligase RTCB in complex with human PYROXD1.
Components
  • Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1
  • RNA-splicing ligase RtcB homolog
KeywordsLIGASE / tRNA ligase / PYROXD1 / RTCB / Flavoprotein
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 / NAD(P)H oxidase H2O2-forming activity / vinculin binding / tRNA processing in the nucleus / placenta development / sarcomere ...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 / NAD(P)H oxidase H2O2-forming activity / vinculin binding / tRNA processing in the nucleus / placenta development / sarcomere / nuclear envelope / cellular response to oxidative stress / in utero embryonic development / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / GTP binding / RNA binding / nucleoplasm / metal ion binding / nucleus / cytoplasm / cytosol
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
DIHYDROFLAVINE-ADENINE DINUCLEOTIDE / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1 / RNA-splicing ligase RtcB homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLoeff, L. / Kroupova, A. / Asanovic, I. / Boneberg, F. / Pfleiderer, M.M. / Ferdigg, A. / Ackle, F. / Martinez, J. / Jinek, M.
Funding support Switzerland, 1items
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, M.M. / Ferdigg, A. / Ackle, F. / Martinez, J. / Jinek, M.
History
DepositionApr 14, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 7, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update
Revision 1.1May 7, 2025Data 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 / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-splicing ligase RtcB homolog
B: Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,0736
Polymers111,5732
Non-polymers1,5004
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry, complex assembly was verified by size exclusion chromatography, co-precipitation and mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein RNA-splicing ligase RtcB homolog / 3'-phosphate/5'-hydroxy nucleic acid ligase


Mass: 55556.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RTCB, C22orf28, HSPC117 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9Y3I0, 3'-phosphate/5'-hydroxy nucleic acid ligase
#2: Protein Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1


Mass: 56016.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PYROXD1 / Production host: Escherichia coli (E. coli)
References: UniProt: Q8WU10, Oxidoreductases; Acting on a sulfur group of donors; With NAD+ or NADP+ as acceptor
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
#5: Chemical ChemComp-FDA / DIHYDROFLAVINE-ADENINE DINUCLEOTIDE


Mass: 787.566 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H35N9O15P2 / Feature type: SUBJECT OF INVESTIGATION
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: Cryo-EM structure of human RTCB in complex with human PYROXD1.
Type: COMPLEX / Details: In vitro reconstituted protein complex / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
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
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaCl1
35 mMMagnesium chlorideMgCl21
40.5 mMTCEPC9H15O6P1
50.5 mMNADHC21H27N7O14P21
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277.15 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: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real images
1164.592GATAN K3 (6k x 4k)15114
2166.459GATAN K3 (6k x 4k)19302

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Processing

EM software
IDNameVersionCategory
2cryoSPARC3.3.2image acquisition
4cryoSPARC2.3.3CTF correction
7Coot0.9.2model fitting
9PHENIXmodel refinement
10cryoSPARC2.3.3initial Euler assignment
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 288325 / Symmetry type: POINT

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