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- PDB-8ide: Structure of an ancient TsaD-TsaC-SUA5-TcdA modular enzyme (TsaN) -

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Basic information

Entry
Database: PDB / ID: 8ide
TitleStructure of an ancient TsaD-TsaC-SUA5-TcdA modular enzyme (TsaN)
ComponentsN(6)-L-threonylcarbamoyladenine synthase
KeywordsVIRAL PROTEIN / Pandovirus salinus / TsaN / modular enzyme / TC-AMP / t6A / ct6A
Function / homology
Function and homology information


ubiquitin-like modifier activating enzyme activity / N6-L-threonylcarbamoyladenine synthase / N(6)-L-threonylcarbamoyladenine synthase activity / tRNA processing / double-stranded RNA binding / metal ion binding
Similarity search - Function
Threonylcarbamoyl-AMP synthase, C-terminal domain / Threonylcarbamoyl-AMP synthase, C-terminal domain superfamily / Threonylcarbamoyl-AMP synthase, C-terminal domain / Threonylcarbamoyl-AMP synthase-like domain / Telomere recombination / YrdC-like domain profile. / Kae1/TsaD family / Gcp-like domain / tRNA N6-adenosine threonylcarbamoyltransferase / DHBP synthase RibB-like alpha/beta domain superfamily ...Threonylcarbamoyl-AMP synthase, C-terminal domain / Threonylcarbamoyl-AMP synthase, C-terminal domain superfamily / Threonylcarbamoyl-AMP synthase, C-terminal domain / Threonylcarbamoyl-AMP synthase-like domain / Telomere recombination / YrdC-like domain profile. / Kae1/TsaD family / Gcp-like domain / tRNA N6-adenosine threonylcarbamoyltransferase / DHBP synthase RibB-like alpha/beta domain superfamily / Ubiquitin-activating enzyme / THIF-type NAD/FAD binding fold / ThiF family / ATPase, nucleotide binding domain
Similarity search - Domain/homology
: / N(6)-L-threonylcarbamoyladenine synthase
Similarity search - Component
Biological speciesPandoravirus salinus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsZhang, Z.L. / Jin, M.Q. / Yu, Z.J. / Chen, W. / Wang, X.L. / Lei, D.S. / Zhang, W.H.
Funding support China, 5items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32000847 China
National Natural Science Foundation of China (NSFC)32171300 China
Other government20JR10RA618
Other governmentlzujbky-2021-ct05
Other government2022021zr46
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structure-function analysis of an ancient TsaD-TsaC-SUA5-TcdA modular enzyme reveals a prototype of tRNA t6A and ct6A synthetases.
Authors: Mengqi Jin / Zelin Zhang / Zhijiang Yu / Wei Chen / Xiaolei Wang / Dongsheng Lei / Wenhua Zhang /
Abstract: N 6-threonylcarbamoyladenosine (t6A) is a post-transcriptional modification found uniquely at position 37 of tRNAs that decipher ANN-codons in the three domains of life. tRNA t6A plays a pivotal role ...N 6-threonylcarbamoyladenosine (t6A) is a post-transcriptional modification found uniquely at position 37 of tRNAs that decipher ANN-codons in the three domains of life. tRNA t6A plays a pivotal role in promoting translational fidelity and maintaining protein homeostasis. The biosynthesis of tRNA t6A requires members from two evolutionarily conserved protein families TsaC/Sua5 and TsaD/Kae1/Qri7, and a varying number of auxiliary proteins. Furthermore, tRNA t6A is modified into a cyclic hydantoin form of t6A (ct6A) by TcdA in bacteria. In this work, we have identified a TsaD-TsaC-SUA5-TcdA modular protein (TsaN) from Pandoraviruses and determined a 3.2 Å resolution cryo-EM structure of P. salinus TsaN. The four domains of TsaN share strong structural similarities with TsaD/Kae1/Qri7 proteins, TsaC/Sua5 proteins, and Escherichia coli TcdA. TsaN catalyzes the formation of threonylcarbamoyladenylate (TC-AMP) using L-threonine, HCO3- and ATP, but does not participate further in tRNA t6A biosynthesis. We report for the first time that TsaN catalyzes a tRNA-independent threonylcarbamoyl modification of adenosine phosphates, leading to t6ADP and t6ATP. Moreover, TsaN is also active in catalyzing tRNA-independent conversion of t6A nucleoside to ct6A. Our results imply that TsaN from Pandoraviruses might be a prototype of the tRNA t6A- and ct6A-modifying enzymes in some cellular organisms.
History
DepositionFeb 13, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: N(6)-L-threonylcarbamoyladenine synthase
B: N(6)-L-threonylcarbamoyladenine synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)211,9823
Polymers211,9272
Non-polymers551
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein N(6)-L-threonylcarbamoyladenine synthase


Mass: 105963.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pandoravirus salinus / Gene: psal_cds_1280 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A291ATS8
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
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: TsaN (TsaD-TsaC-SUA5-TcdA) / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.2095 MDa / Experimental value: YES
Source (natural)Organism: Pandoravirus salinus
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 700000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152436 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeSource nameType
14YDU

4ydu

14YDUPDBexperimental model
26F89

6f89

16F89PDBexperimental model
34RDI

4rdi

14RDIPDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 107.02 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038520
ELECTRON MICROSCOPYf_angle_d0.726111636
ELECTRON MICROSCOPYf_chiral_restr0.04411419
ELECTRON MICROSCOPYf_plane_restr0.00481535
ELECTRON MICROSCOPYf_dihedral_angle_d13.14282979

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