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- EMDB-52744: Cryo-EM structure of the Danio rerio tRNA ligase complex -

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Basic information

Entry
Database: EMDB / ID: EMD-52744
TitleCryo-EM structure of the Danio rerio tRNA ligase complex
Map dataMap sharpened locally in cryoSPARC with Local Filtering tool
Sample
  • Complex: tRNA ligase complex from D. rerio
    • Protein or peptide: ATP-dependent RNA helicase
    • Protein or peptide: RNA-splicing ligase RtcB homolog
    • Protein or peptide: Family with sequence similarity 98, member B
    • Protein or peptide: RNA transcription, translation and transport factor protein
    • Protein or peptide: Ashwin
KeywordstRNA ligase complex / RTCB / tRNA / tRNA maturation / tRNA splicing / LIGASE / 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 / cytoplasmic stress granule / 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 / cytoplasm / cytosol
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)
Methodsingle 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, 1 items
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, 2025-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52744.png

Downloads & links

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Map

FileDownload / File: emd_52744.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened locally in cryoSPARC with Local Filtering tool
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 256 pix.
= 215.04 Å		0.84 Å/pix.
x 256 pix.
= 215.04 Å		0.84 Å/pix.
x 256 pix.
= 215.04 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.8446383 - 2.4197993
Average (Standard dev.)0.0015807287 (±0.052890323)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 215.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52744_msk_1.map
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Mask #2

Fileemd_52744_msk_2.map
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Additional map: Unsharpened map

Fileemd_52744_additional_1.map
AnnotationUnsharpened map
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Half map: half map B

Fileemd_52744_half_map_1.map
Annotationhalf map B
Projections & Slices
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Half map: half map A

Fileemd_52744_half_map_2.map
Annotationhalf map A
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Sample components

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Entire : tRNA ligase complex from D. rerio

EntireName: tRNA ligase complex from D. rerio
Components
  • Complex: tRNA ligase complex from D. rerio
    • Protein or peptide: ATP-dependent RNA helicase
    • Protein or peptide: RNA-splicing ligase RtcB homolog
    • Protein or peptide: Family with sequence similarity 98, member B
    • Protein or peptide: RNA transcription, translation and transport factor protein
    • Protein or peptide: Ashwin

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Supramolecule #1: tRNA ligase complex from D. rerio

SupramoleculeName: tRNA ligase complex from D. rerio / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 133 KDa

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Macromolecule #1: ATP-dependent RNA helicase

MacromoleculeName: ATP-dependent RNA helicase / type: protein_or_peptide / ID: 1 / Details: truncated variant (aa 693-740) / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 5.349087 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths)
SequenceString:
RRATGGGLYK GHVDILAPTV QELANLEREA QTSFLHLGYL PNQLFKAF

UniProtKB: ATP-dependent RNA helicase

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Macromolecule #2: RNA-splicing ligase RtcB homolog

MacromoleculeName: RNA-splicing ligase RtcB homolog / type: protein_or_peptide / ID: 2 / Details: HIS-TEV-RTCB / Number of copies: 1 / Enantiomer: LEVO / EC number: 3'-phosphate/5'-hydroxy nucleic acid ligase
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 57.22525 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths)
SequenceString: MHHHHHHENL YFQGMSRSYN DELQYLDKIH KNCWRIKKGF VPNMLVEGVF YVNDPLEKLM FEELRNACRG GGFGGFLPAM KQIGNVAAL PGIVHRSIGL PDVHSGYGFA IGNMAAFDME NPDAVVSPGG VGFDINCGVR LLRTNLDEGD VQPVKEQLAQ S LFDHIPVG ...String:
MHHHHHHENL YFQGMSRSYN DELQYLDKIH KNCWRIKKGF VPNMLVEGVF YVNDPLEKLM FEELRNACRG GGFGGFLPAM KQIGNVAAL PGIVHRSIGL PDVHSGYGFA IGNMAAFDME NPDAVVSPGG VGFDINCGVR LLRTNLDEGD VQPVKEQLAQ S LFDHIPVG VGSKGVIPMG AKDLEEALEM GVDWSLREGY AWAEDKEHCE EYGRMLQADP NKVSSKAKKR GLPQLGTLGA GN HYAEIQV VDEIYNDYAA KKMGIDHKGQ VCVMIHSGSR GLGHQVATDA LVAMEKAMKR DRITVNDRQL ACARITSEEG QDY LKGMAA AGNYAWVNRS SMTFLTRQAF SKVFSTTPDD LDMHVIYDVS HNIAKVEEHM VDGRQKTLLV HRKGSTRAFP PHHP LIPVD YQLTGQPVLI GGTMGTCSYV LTGTEQGMTE TFGTTCHGAG RALSRAKSRR NLDFQDVLDK LADMGIAIRV ASPKL VMEE APESYKNVTD VVNTCHDAGI SKKAIKLRPI AVIKG

UniProtKB: RNA-splicing ligase RtcB homolog

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Macromolecule #3: Family with sequence similarity 98, member B

MacromoleculeName: Family with sequence similarity 98, member B / type: protein_or_peptide / ID: 3 / Details: truncated variant (aa 1-296) / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 33.196734 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths)
SequenceString: MESDILDILE QLGYDGPLAE EACLLAECGR GFSSSEYVNL LTWLTKQLTQ FTETHTQDEI ITADPLDVSR LLKDCCCPYE GLASRLANG DVKDTRDHLK IILFVSSELQ SAQLLLSKTL RDAEEREMRS CSPLQDLSVI CHTLTLPDPA GRDPTDTFTD I QTQVNVLL ...String:
MESDILDILE QLGYDGPLAE EACLLAECGR GFSSSEYVNL LTWLTKQLTQ FTETHTQDEI ITADPLDVSR LLKDCCCPYE GLASRLANG DVKDTRDHLK IILFVSSELQ SAQLLLSKTL RDAEEREMRS CSPLQDLSVI CHTLTLPDPA GRDPTDTFTD I QTQVNVLL EKLPETHIGA PALQRSISAE QWEELEKINS TLSAEYECRR RMLIKRLDVT VQSFSWSDRA KVKIDQMARA YQ PKRHSLS VRSSVSLAHL LAARRDICNM VKTSSGSSRQ NTSCAVNRIL MGRVPDRGG

UniProtKB: Family with sequence similarity 98, member B

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Macromolecule #4: RNA transcription, translation and transport factor protein

MacromoleculeName: RNA transcription, translation and transport factor protein
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 27.911844 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths)
SequenceString: MFRRKLTALE YHNPTGFDCK DETEFRNFIV WLEDQKIRHY KIEDRGNLRN IPSSDWPKYF EKYLQDVNCP FSVQERQETV DWLLGLAVR FEYGDNVEKY RNCKPVTETN DVQKSADPLI NLDSNNPDFK AGVLALANLL KIQRHDDYLV MLKAIKILVQ E RLTPDAIA ...String:
MFRRKLTALE YHNPTGFDCK DETEFRNFIV WLEDQKIRHY KIEDRGNLRN IPSSDWPKYF EKYLQDVNCP FSVQERQETV DWLLGLAVR FEYGDNVEKY RNCKPVTETN DVQKSADPLI NLDSNNPDFK AGVLALANLL KIQRHDDYLV MLKAIKILVQ E RLTPDAIA KASQAKEGLP VTLDKHILGF DTGDATLNEA AQILRLLHIE ELRELQTKIN EAIVAVQAII ADPKTDHRLG KV GR

UniProtKB: RNA transcription, translation and transport factor protein

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Macromolecule #5: Ashwin

MacromoleculeName: Ashwin / type: protein_or_peptide / ID: 5 / Details: truncated variant (aa 1-83) / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 9.706074 KDa
Recombinant expressionOrganism: Spodoptera (butterflies/moths)
SequenceString:
MASHRTDRTK NPTSNGDVSK VDLLLHPELL SQEFIQLMLQ ERNIAVSDPE DRDRLTGLYL QHVIPLPQRE LPRSRWGKRM EKS

UniProtKB: Ashwin

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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

Concentration0.97 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
150.0 mMNaClsodium chloride
1.0 mMDTTDithiothreitol
2.0 mMMnCl2Manganese(II) chloride
2.0 mMGTPGuanosine-5'-triphosphate
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Detailsstage tilt 20 deg
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 9942 / Average electron dose: 41.09 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 13964718
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 179455
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 5
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL
Output model

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

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