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- EMDB-26856: Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG -

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Basic information

Entry
Database: EMDB / ID: EMD-26856
TitleHuman tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG
Map data
Sample
  • Complex: Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen34
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen15
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen2
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen54
    • RNA: RNA (78-MER)
  • Ligand: MAGNESIUM ION
Keywordssplicing endonuclease / pre-tRNA / TSEN / EndA / SPLICING-RNA complex
Function / homology
Function and homology information


tRNA-intron endonuclease complex / tRNA-type intron splice site recognition and cleavage / tRNA-intron lyase / tRNA-intron endonuclease activity / tRNA splicing, via endonucleolytic cleavage and ligation / tRNA processing in the nucleus / mRNA processing / nucleic acid binding / lyase activity / centrosome ...tRNA-intron endonuclease complex / tRNA-type intron splice site recognition and cleavage / tRNA-intron lyase / tRNA-intron endonuclease activity / tRNA splicing, via endonucleolytic cleavage and ligation / tRNA processing in the nucleus / mRNA processing / nucleic acid binding / lyase activity / centrosome / nucleolus / nucleoplasm / cytosol
Similarity search - Function
tRNA-splicing endonuclease, SEN2 subunit / tRNA-splicing endonuclease, subunit Sen54, N-terminal / tRNA-splicing endonuclease, subunit Sen54 / tRNA-splicing endonuclease subunit sen54 N-term / tRNA-splicing endonuclease, SEN34 subunit / tRNA-splicing endonuclease subunit Sen15 / Sen15 protein / tRNA intron endonuclease, N-terminal / tRNA intron endonuclease, N-terminal domain / tRNA-splicing endonuclease ...tRNA-splicing endonuclease, SEN2 subunit / tRNA-splicing endonuclease, subunit Sen54, N-terminal / tRNA-splicing endonuclease, subunit Sen54 / tRNA-splicing endonuclease subunit sen54 N-term / tRNA-splicing endonuclease, SEN34 subunit / tRNA-splicing endonuclease subunit Sen15 / Sen15 protein / tRNA intron endonuclease, N-terminal / tRNA intron endonuclease, N-terminal domain / tRNA-splicing endonuclease / tRNA intron endonuclease, catalytic domain-like / tRNA intron endonuclease, catalytic C-terminal domain / tRNA intron endonuclease, catalytic domain-like superfamily / tRNA endonuclease-like domain superfamily
Similarity search - Domain/homology
tRNA-splicing endonuclease subunit Sen54 / tRNA-splicing endonuclease subunit Sen2 / tRNA-splicing endonuclease subunit Sen15 / tRNA-splicing endonuclease subunit Sen34
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsStanley RE / Hayne CK
Funding support United States, 8 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIA ES103247 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIC ES102488 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIC ES103206 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIC ES102487 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZI ES043010 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIC ES103326 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1K99-GM143534 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM136435 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex.
Authors: Cassandra K Hayne / Kevin John U Butay / Zachary D Stewart / Juno M Krahn / Lalith Perera / Jason G Williams / Robert M Petrovitch / Leesa J Deterding / A Gregory Matera / Mario J Borgnia / Robin E Stanley /
Abstract: Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is ...Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential, and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). Here, we report cryo-electron microscopy structures of the human TSEN-pre-tRNA complex. These structures reveal the overall architecture of the complex and the extensive tRNA binding interfaces. The structures share homology with archaeal TSENs but contain additional features important for pre-tRNA recognition. The TSEN54 subunit functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits. Finally, the TSEN structures enable visualization of the molecular environments of PCH-causing missense mutations, providing insight into the mechanism of pre-tRNA splicing and PCH.
History
DepositionMay 5, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26856.png

Downloads & links

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Map

FileDownload / File: emd_26856.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.932 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.9692672 - 3.1446855
Average (Standard dev.)0.000710873 (±0.051796015)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 298.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_26856_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26856_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG

EntireName: Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG
Components
  • Complex: Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen34
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen15
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen2
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen54
    • RNA: RNA (78-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG

SupramoleculeName: Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 203 KDa

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Macromolecule #1: tRNA-splicing endonuclease subunit Sen34

MacromoleculeName: tRNA-splicing endonuclease subunit Sen34 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: tRNA-intron lyase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.941316 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASWSHPQFE KGGGARGGSG GGSWSHPQFE KGFDYKDDDD KGTMLVVEVA NGRSLVWGAE AVQALRERLG VGGRTVGALP RGPRQNSRL GLPLLLMPEE ARLLAEIGAV TLVSAPRPDS RHHSLALTSF KRQQEESFQE QSALAAEARE TRRQELLEKI T EGQAAKKQ ...String:
MASWSHPQFE KGGGARGGSG GGSWSHPQFE KGFDYKDDDD KGTMLVVEVA NGRSLVWGAE AVQALRERLG VGGRTVGALP RGPRQNSRL GLPLLLMPEE ARLLAEIGAV TLVSAPRPDS RHHSLALTSF KRQQEESFQE QSALAAEARE TRRQELLEKI T EGQAAKKQ KLEQASGASS SQEAGSSQAA KEDETSDGQA SGEQEEAGPS SSQAGPSNGV APLPRSALLV QLATARPRPV KA RPLDWRV QSKDWPHAGR PAHELRYSIY RDLWERGFFL SAAGKFGGDF LVAPGDPLRF AAHYIAQCWA PEDTIPLQDL VAA GRLGTS VRATLLLCSP QPDGKVVYTS LQWASL

UniProtKB: tRNA-splicing endonuclease subunit Sen34

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Macromolecule #2: tRNA-splicing endonuclease subunit Sen15

MacromoleculeName: tRNA-splicing endonuclease subunit Sen15 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 19.996619 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MEERGDSEPT PGCSGLGPGG VRGFGDGGGA PSWAPEDAWM GTHPKYLEMM ELDIGDATQV YVAFLVYLDL MESKSWHEVN CVGLPELQL ICLVGTEIEG EGLQTVVPTP ITASLSHNRI REILKASRKL QGDPDLPMSF TLAIVESDST IVYYKLTDGF M LPDPQNIS LRRGPEQKLI SEEDL

UniProtKB: tRNA-splicing endonuclease subunit Sen15

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Macromolecule #3: tRNA-splicing endonuclease subunit Sen2

MacromoleculeName: tRNA-splicing endonuclease subunit Sen2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: tRNA-intron lyase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.352961 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAEAVFHAPK RKRRVYETYE SPLPIPFGQD HGPLKEFKIF RAEMINNNVI VRNAEDIEQL YGKGYFGKGI LSRSRPSFTI SDPKLVAKW KDMKTNMPII TSKRYQHSVE WAAELMRRQG QDESTVRRIL KDYTKPLEHP PVKRNEEAQV HDKLNSGMVS N MEGTAGGE ...String:
MAEAVFHAPK RKRRVYETYE SPLPIPFGQD HGPLKEFKIF RAEMINNNVI VRNAEDIEQL YGKGYFGKGI LSRSRPSFTI SDPKLVAKW KDMKTNMPII TSKRYQHSVE WAAELMRRQG QDESTVRRIL KDYTKPLEHP PVKRNEEAQV HDKLNSGMVS N MEGTAGGE RPSVVNGDSG KSGGVGDPRE PLGCLQEGSG CHPTTESFEK SVREDASPLP HVCCCKQDAL ILQRGLHHED GS QHIGLLH PGDRGPDHEY VLVEEAECAM SEREAAPNEE LVQRNRLICR RNPYRIFEYL QLSLEEAFFL VYALGCLSIY YEK EPLTIV KLWKAFTVVQ PTFRTTYMAY HYFRSKGWVP KVGLKYGTDL LLARKGPPFY AASYSVIIEL VDDHFEGSLR RPLS WKSLA ALSRVSVNVS AELMLCYLIK PSTMTDKEME SPECMKRIKV QEVILSRWVS SRERSDQDDL DYKDDDDKGF WSHPQ FEK

UniProtKB: tRNA-splicing endonuclease subunit Sen2

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Macromolecule #4: tRNA-splicing endonuclease subunit Sen54

MacromoleculeName: tRNA-splicing endonuclease subunit Sen54 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 61.863391 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEPEPEPAAV EVPAGRVLSA RELFAARSRS QKLPQRSHGP KDFLPDGSAA QAERLRRCRE ELWQLLAEQR VERLGSLVAA EWRPEEGFV ELKSPAGKFW QTMGFSEQGR QRLHPEEALY LLECGSIHLF HQDLPLSIQE AYQLLLTDHT VTFLQYQVFS H LKRLGYVV ...String:
MEPEPEPAAV EVPAGRVLSA RELFAARSRS QKLPQRSHGP KDFLPDGSAA QAERLRRCRE ELWQLLAEQR VERLGSLVAA EWRPEEGFV ELKSPAGKFW QTMGFSEQGR QRLHPEEALY LLECGSIHLF HQDLPLSIQE AYQLLLTDHT VTFLQYQVFS H LKRLGYVV RRFQPSSVLS PYERQLNLDA SVQHLEDGDG KRKRSSSSPR SINKKAKALD NSLQPKSLAA SSPPPCSQPS QC PEEKPQE SSPMKGPGGP FQLLGSLGPS PGPAREGVGC SWESGRAENG VTGAGKRRWN FEQISFPNMA SDSRHTLLRA PAP ELLPAN VAGRETDAES WCQKLNQRKE KLSRREREHH AEAAQFQEDV NADPEVQRCS SWREYKELLQ RRQVQRSQRR APHL WGQPV TPLLSPGQAS SPAVVLQHIS VLQTTHLPDG GARLLEKSGG LEIIFDVYQA DAVATFRKNN PGKPYARMCI SGFDE PVPD LCSLKRLSYQ SGDVPLIFAL VDHGDISFYS FRDFTLPQDV GHQAYVEQKL ISEEDLNSAV DHHHHHH

UniProtKB: tRNA-splicing endonuclease subunit Sen54

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Macromolecule #5: RNA (78-MER)

MacromoleculeName: RNA (78-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 28.440879 KDa
SequenceString:
GGCUCUGUGG CGCAAUGGAU AGCGCAUUGG ACUUCUAGUG ACGAAUAGAG CAAUUCAAAG GUUGUGGGUU CGAAUCCCAC CAGAGUCG

GENBANK: GENBANK: Z26635.1

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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: 8
GridModel: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TALOS ARCTICA
Image recordingImage recording ID: 1 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 45000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Electron microscopy #1~

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Image recordingImage recording ID: 2 / Film 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 / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID2
Particle selectionNumber selected: 570104
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6z9u


Details: Used alphafold models and 6Z9U to dock in prior to refinements
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 152031
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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