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- PDB-9i8v: Cryo-EM structure of the Danio rerio tRNA ligase complex -

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Basic information

Entry
Database: PDB / ID: 9i8v
TitleCryo-EM structure of the Danio rerio tRNA ligase complex
Components
  • ATP-dependent RNA helicase
  • Ashwin
  • Family with sequence similarity 98, member B
  • RNA transcription, translation and transport factor protein
  • RNA-splicing ligase RtcB homolog
KeywordsLIGASE / tRNA ligase complex / RTCB / tRNA / tRNA maturation / tRNA splicing / RNA BINDING PROTEIN
Function / homology
Function and homology information


tRNA-splicing ligase complex / 3'-phosphate/5'-hydroxy nucleic acid ligase / RNA ligase (GTP) activity / tRNA splicing, via endonucleolytic cleavage and ligation / embryonic morphogenesis / exonuclease activity / tRNA processing / RNA polymerase II complex binding / negative regulation of protein kinase activity / mRNA processing ...tRNA-splicing ligase complex / 3'-phosphate/5'-hydroxy nucleic acid ligase / RNA ligase (GTP) activity / tRNA splicing, via endonucleolytic cleavage and ligation / embryonic morphogenesis / exonuclease activity / tRNA processing / RNA polymerase II complex binding / negative regulation of protein kinase activity / mRNA processing / mitotic spindle / RNA helicase activity / RNA helicase / centrosome / GTP binding / perinuclear region of cytoplasm / positive regulation of transcription by RNA polymerase II / mitochondrion / DNA binding / RNA binding / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
FAM98 / Ashwin / Protein of unknown function (DUF2465) / Developmental protein / RNA transcription, translation and transport factor protein / RNA transcription, translation and transport factor protein / RNA-splicing ligase RtcB homologue, eukaryotic / Uncharacterized protein family UPF0027 signature. / RNA-splicing ligase, RtcB / tRNA-splicing ligase RtcB-like superfamily ...FAM98 / Ashwin / Protein of unknown function (DUF2465) / Developmental protein / RNA transcription, translation and transport factor protein / RNA transcription, translation and transport factor protein / 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 / RNA helicase, DEAD-box type, Q motif / DEAD-box RNA helicase Q motif profile. / SPRY domain / B30.2/SPRY domain / B30.2/SPRY domain profile. / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / SPRY domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Concanavalin A-like lectin/glucanase domain superfamily / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Family with sequence similarity 98, member B / ATP-dependent RNA helicase / Ashwin / RNA-splicing ligase RtcB homolog / RNA transcription, translation and transport factor protein
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsChamera, S. / Zajko, W. / Czarnocki-Cieciura, M. / Jaciuk, M. / Koziej, L. / Nowak, J. / Wycisk, K. / Sroka, M. / Chramiec-Glabik, A. / Smietanski, M. ...Chamera, S. / Zajko, W. / Czarnocki-Cieciura, M. / Jaciuk, M. / Koziej, L. / Nowak, J. / Wycisk, K. / Sroka, M. / Chramiec-Glabik, A. / Smietanski, M. / Golebiowski, F. / Warminski, M. / Jemielity, J. / Glatt, S. / Nowotny, M.
Funding support Poland, 1items
OrganizationGrant numberCountry
Polish National Science Centre2019/33/B/NZ1/02839 Poland
CitationJournal: J Biol Chem / Year: 2025
Title: Structural and biochemical characterization of the 3'-5' tRNA splicing ligases.
Authors: Sebastian Chamera / Weronika Zajko / Mariusz Czarnocki-Cieciura / Marcin Jaciuk / Łukasz Koziej / Jakub Nowak / Krzysztof Wycisk / Małgorzata Sroka / Andrzej Chramiec-Głąbik / Mirosław ...Authors: Sebastian Chamera / Weronika Zajko / Mariusz Czarnocki-Cieciura / Marcin Jaciuk / Łukasz Koziej / Jakub Nowak / Krzysztof Wycisk / Małgorzata Sroka / Andrzej Chramiec-Głąbik / Mirosław Śmietański / Filip Gołębiowski / Marcin Warmiński / Jacek Jemielity / Sebastian Glatt / Marcin Nowotny /
Abstract: In archaea and metazoa, tRNA exons are ligated by the RNA ligases RtcB and RTCB, respectively. The metazoan RTCB forms a stable complex with four additional subunits, DDX1, FAM98B, CGI99, and ASHWIN. ...In archaea and metazoa, tRNA exons are ligated by the RNA ligases RtcB and RTCB, respectively. The metazoan RTCB forms a stable complex with four additional subunits, DDX1, FAM98B, CGI99, and ASHWIN. The role and assembly of these four components remain elusive. Furthermore, we lack structural information of how RNA substrates are recognized by 3'-5' tRNA ligases. Here, we use thiol-based chemical crosslinking to confirm the involvement of specific residues of RtcB in RNA binding, and we present a cryo-EM structure of the purified five-subunit Danio rerio tRNA ligase complex. The structure implies that the DDX1 helicase module is mobile and can modulate the activity of RTCB. Taken together, the presented results enhance our mechanistic understanding of RNA binding by 3'-5' tRNA splicing ligases and architecture of the metazoan tRNA ligase complex.
History
DepositionFeb 6, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
Revision 1.1May 21, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: ATP-dependent RNA helicase
C: RNA-splicing ligase RtcB homolog
D: Family with sequence similarity 98, member B
E: RNA transcription, translation and transport factor protein
F: Ashwin


Theoretical massNumber of molelcules
Total (without water)133,3895
Polymers133,3895
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein/peptide ATP-dependent RNA helicase


Mass: 5349.087 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: truncated variant (aa 693-740) / Source: (gene. exp.) Danio rerio (zebrafish) / Gene: ddx1, si:ch211-214k9.2, si:dkey-161f12.1 / Production host: Spodoptera (butterflies/moths) / References: UniProt: F1R2L8, RNA helicase
#2: Protein RNA-splicing ligase RtcB homolog / 3'-phosphate/5'-hydroxy nucleic acid ligase


Mass: 57225.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: HIS-TEV-RTCB / Source: (gene. exp.) Danio rerio (zebrafish) / Gene: rtcb, zgc:76871 / Production host: Spodoptera (butterflies/moths)
References: UniProt: Q6NZS4, 3'-phosphate/5'-hydroxy nucleic acid ligase
#3: Protein Family with sequence similarity 98, member B / Protein FAM98B


Mass: 33196.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: truncated variant (aa 1-296) / Source: (gene. exp.) Danio rerio (zebrafish) / Gene: fam98b, zgc:158382 / Production host: Spodoptera (butterflies/moths) / References: UniProt: A0PJS3
#4: Protein RNA transcription, translation and transport factor protein


Mass: 27911.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: rtraf, zgc:56576 / Production host: Spodoptera (butterflies/moths) / References: UniProt: Q7ZUH1
#5: Protein Ashwin


Mass: 9706.074 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: truncated variant (aa 1-83) / Source: (gene. exp.) Danio rerio (zebrafish) / Gene: im:6907446, si:dkeyp-118h3.6 / Production host: Spodoptera (butterflies/moths) / References: UniProt: Q32LR5
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: tRNA ligase complex from D. rerio / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.133 MDa / Experimental value: NO
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Spodoptera (butterflies/moths)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESHEPES1
2150 mMsodium chlorideNaCl1
31 mMDithiothreitolDTT1
42 mMManganese(II) chlorideMnCl21
52 mMGuanosine-5'-triphosphateGTP1
SpecimenConc.: 0.97 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS / Details: stage tilt 20 deg
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: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 41.09 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9942

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 13964718
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179455 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 75.17 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00297869
ELECTRON MICROSCOPYf_angle_d0.513410665
ELECTRON MICROSCOPYf_chiral_restr0.03871231
ELECTRON MICROSCOPYf_plane_restr0.0041374
ELECTRON MICROSCOPYf_dihedral_angle_d3.64241087

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