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- EMDB-35365: Structure of an ancient TsaD-TsaC-SUA5-TcdA modular enzyme (TsaN) -

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Basic information

Entry
Database: EMDB / ID: EMD-35365
TitleStructure of an ancient TsaD-TsaC-SUA5-TcdA modular enzyme (TsaN)
Map data
Sample
  • Organelle or cellular component: TsaN (TsaD-TsaC-SUA5-TcdA)
    • Protein or peptide: N(6)-L-threonylcarbamoyladenine synthase
  • Ligand: MANGANESE (II) ION
KeywordsPandovirus salinus / TsaN / modular enzyme / TC-AMP / t6A / ct6A / VIRAL PROTEIN
Function / homology
Function and homology information


N6-L-threonylcarbamoyladenine synthase / N(6)-L-threonylcarbamoyladenine synthase activity / ubiquitin-like modifier activating enzyme 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 / THIF-type NAD/FAD binding fold / ThiF family / Ubiquitin-activating enzyme / ATPase, nucleotide binding domain
Similarity search - Domain/homology
N(6)-L-threonylcarbamoyladenine synthase
Similarity search - Component
Biological speciesPandoravirus salinus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsZhang ZL / Jin MQ / Yu ZJ / Chen W / Wang XL / Lei DS / Zhang WH
Funding support China, 5 items
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, 2023-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35365.png

Downloads & links

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Map

FileDownload / File: emd_35365.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å		0.82 Å/pix.
x 512 pix.
= 419.84 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.359
Minimum - Maximum-0.56336886 - 1.2011262
Average (Standard dev.)-0.0005517014 (±0.025716236)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 419.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35365_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35365_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : TsaN (TsaD-TsaC-SUA5-TcdA)

EntireName: TsaN (TsaD-TsaC-SUA5-TcdA)
Components
  • Organelle or cellular component: TsaN (TsaD-TsaC-SUA5-TcdA)
    • Protein or peptide: N(6)-L-threonylcarbamoyladenine synthase
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: TsaN (TsaD-TsaC-SUA5-TcdA)

SupramoleculeName: TsaN (TsaD-TsaC-SUA5-TcdA) / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pandoravirus salinus
Molecular weightTheoretical: 209.5 KDa

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

MacromoleculeName: N(6)-L-threonylcarbamoyladenine synthase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pandoravirus salinus
Molecular weightTheoretical: 105.963406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSVRAQPHM SGRAPDAANV AGAVRTSAGM CATGANFERR VTILGIESSC DDTGVAVLQV GGNAPPAVLA HEAVTSWAHH ARQADINKE HAAAVHRETV APLVDQAMAA SGVGWDAIDA IAVTVGPGMM GGLMAGVDEA VRLAALHGKP LVPVNHLEGH A LVAGVCTR ...String:
MSSVRAQPHM SGRAPDAANV AGAVRTSAGM CATGANFERR VTILGIESSC DDTGVAVLQV GGNAPPAVLA HEAVTSWAHH ARQADINKE HAAAVHRETV APLVDQAMAA SGVGWDAIDA IAVTVGPGMM GGLMAGVDEA VRLAALHGKP LVPVNHLEGH A LVAGVCTR QLCFPFLVLL ASGGSCQLVL ARDLGDYRRL GQTLDCAPGQ ALDAVARALA LDLGASGSGG RAIELAAKNA RT DAGDDRI GDDAWPDGCD FAFGGLRDRA VALARKSLAG EADDIAKRVQ ALIVDQLVSR TVRAIEWCRA HVADPTALVV AGG VAANTC LRESLQRAIG SVDLVCPPPR LCTDNGVMIA HAGALHYLHR PDAFACGPTH VCLQHEWHLG VDVSECVRAD RPVP QVAAI HASIKSDVAD AARALCRGEL VAFPTETVYG LGADAASDEA VQRIFDAKGR PSNNPIIVHV ASKEQFYRIA GHDLD AALR ARCERLMDEF WPGPLTLLVP NGGEKLSPLV TCGLPVVGLR MPDNATAIDL IRRAGVGVAA PSANKSGRPS PTCAQH VAA DLVGERIWGV LDGRGSTYGI ESTVLDVATV SIYREGPVTA DDISRALDGA PVDVSSGRKE LAAGEAPKAP GMLYRHY AP DTDVTVVHGT LGFLNATVRS MRDRGLRVGV IAPYGDAIDA RASKVWYCMR HGDATDVMGS LGANLYAALR GLDLPDVD V ILVRAVPDSR TGGAVMERLA KASQGSRLIE PAMTARLERM IGADVVQRIA RGRVLVCGLG GAGAPLVDMA VRAGVGRLG LLDPDRVDLS NLVRMPQATL ADVDRRKIDV VAERARAVNP DADLTLLAHR ITPDFDMGAL RAHEYDIIVD AVDDPAGKVA LIKYAVENK LPLISCMGAG NKTDVTQVHR VVDIADADVC LLALETKRLL AKEGITRGVK CVVTQGDHWV FAPQDGHDVI G NWPPCYFM AAAVLLDHVL RVLAGPESVE DHVRGRAVGV STKSGIVAIP

UniProtKB: N(6)-L-threonylcarbamoyladenine synthase

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Macromolecule #2: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MN
Molecular weightTheoretical: 54.938 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

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 700000
Startup model#0 - Type of model: NONE / #1 - Type of model: NONE / #2 - Type of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 152436
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4ydu

source_name: PDB, initial_model_type: experimental model

6f89

source_name: PDB, initial_model_type: experimental model

4rdi

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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