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- EMDB-14923: Structure of the human tRNA splicing endonuclease defines substra... -

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Basic information

Entry
Database: EMDB / ID: EMD-14923
TitleStructure of the human tRNA splicing endonuclease defines substrate recognition
Map datamap
Sample
  • Complex: Human TSEN with pre-tRNA-Arg-TCT
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen34
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen2
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen54
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen15
    • RNA: pre-tRNA Arg TCT 3-2
KeywordsRNP / endonuclease / tRNA / splicing / RNA BINDING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsSekulovski S / Trowitzsch S
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)TR 1711/1-7 Germany
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN.
Authors: Samoil Sekulovski / Lukas Sušac / Lukas S Stelzl / Robert Tampé / Simon Trowitzsch /
Abstract: Heterotetrameric human transfer RNA (tRNA) splicing endonuclease TSEN catalyzes intron excision from precursor tRNAs (pre-tRNAs), utilizing two composite active sites. Mutations in TSEN and its ...Heterotetrameric human transfer RNA (tRNA) splicing endonuclease TSEN catalyzes intron excision from precursor tRNAs (pre-tRNAs), utilizing two composite active sites. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH). Despite the essential function of TSEN, the three-dimensional assembly of TSEN-CLP1, the mechanism of substrate recognition, and the structural consequences of disease mutations are not understood in molecular detail. Here, we present single-particle cryogenic electron microscopy reconstructions of human TSEN with intron-containing pre-tRNAs. TSEN recognizes the body of pre-tRNAs and pre-positions the 3' splice site for cleavage by an intricate protein-RNA interaction network. TSEN subunits exhibit large unstructured regions flexibly tethering CLP1. Disease mutations localize far from the substrate-binding interface and destabilize TSEN. Our work delineates molecular principles of pre-tRNA recognition and cleavage by human TSEN and rationalizes mutations associated with PCH.
History
DepositionMay 5, 2022-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateJun 28, 2023-
Current statusJun 28, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14923.png

Downloads & links

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Map

FileDownload / File: emd_14923.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap
Voxel sizeX=Y=Z: 0.78 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.4498353 - 5.1354985
Average (Standard dev.)0.000059720824 (±0.07333189)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 299.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14923_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_14923_half_map_1.map
Annotationhalf_map_B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_14923_half_map_2.map
Annotationhalf_map_A
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 TSEN with pre-tRNA-Arg-TCT

EntireName: Human TSEN with pre-tRNA-Arg-TCT
Components
  • Complex: Human TSEN with pre-tRNA-Arg-TCT
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen34
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen2
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen54
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen15
    • RNA: pre-tRNA Arg TCT 3-2

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Supramolecule #1: Human TSEN with pre-tRNA-Arg-TCT

SupramoleculeName: Human TSEN with pre-tRNA-Arg-TCT / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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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: 28.805811 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLVVEVANGR SLVWGAEAVQ ALRERLGVGG RTVGALPRGP RQNSRLGLPL LLMPEEARLL AEIGAVTLVS APRPDSRHHS LALTSFKRQ QEESFQEQSA LAAEARETRR QELLEKITEG QGGSGGSGGS GGSRSALLVQ LATARPRPVK ARPLDWRVQS K DWPHAGRP ...String:
MLVVEVANGR SLVWGAEAVQ ALRERLGVGG RTVGALPRGP RQNSRLGLPL LLMPEEARLL AEIGAVTLVS APRPDSRHHS LALTSFKRQ QEESFQEQSA LAAEARETRR QELLEKITEG QGGSGGSGGS GGSRSALLVQ LATARPRPVK ARPLDWRVQS K DWPHAGRP AHELRYSIYR DLWERGFFLS AAGKFGGDFL VYPGDPLRFF AHYIAQCWAP EDTIPLQDLV AAGRLGTSVR KT LLLCSPQ PDGKVVYTSL QWASLQ

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Macromolecule #2: tRNA-splicing endonuclease subunit Sen2

MacromoleculeName: tRNA-splicing endonuclease subunit Sen2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: tRNA-intron lyase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 30.165961 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAEAVFHAPK RKRRVYETYE SPLPIPFGQD HGPLKEFKIF RAEMINNNVI VRNAEDIEQL YGKGYFGKGI LSRSRPSFTI SGGSGGRIF EYLQLSLEEA FFLVYALGCL SIYYEKEPLT IVKLWKAFTV VQPTFRTTYM AYHYFRSKGW VPKVGLKYGT D LLLYRKGP ...String:
MAEAVFHAPK RKRRVYETYE SPLPIPFGQD HGPLKEFKIF RAEMINNNVI VRNAEDIEQL YGKGYFGKGI LSRSRPSFTI SGGSGGRIF EYLQLSLEEA FFLVYALGCL SIYYEKEPLT IVKLWKAFTV VQPTFRTTYM AYHYFRSKGW VPKVGLKYGT D LLLYRKGP PFYFASYSVI IELVDDHFEG SLRRPLSWKS LAALSRVSVN VSKELMLCYL IKPSTMTDKE MESPECMKRI KV QEVILSR WVSSRERSDQ DDL

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Macromolecule #3: tRNA-splicing endonuclease subunit Sen54

MacromoleculeName: tRNA-splicing endonuclease subunit Sen54 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.551711 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 GGSGGSGGSG GSGSVLQTTH LPDGGARLLE KSGGLEIIFD VYQADAVATF RKNNPGKPYA RM CISGFDE PVPDLCSLKR LSYQSGDVPL IFALVDHGDI SFYSFRDFTL PQDVGH

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Macromolecule #4: tRNA-splicing endonuclease subunit Sen15

MacromoleculeName: tRNA-splicing endonuclease subunit Sen15 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.860889 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDEKTTGWRG GHVVEGLAGE LEQLRARLEH HPQGQREPGE NLYFQGMEER GDSEPTPGCS GLGPGGVRGF GDGGGAPSWA PEDAWMGTH PKYLEMMELD IGDATQVYVA FLVYLDLMES KSWHEVNCVG LPELQLICLV GTEIEGEGLQ TVVPTPITAS L SHNRIREI ...String:
MDEKTTGWRG GHVVEGLAGE LEQLRARLEH HPQGQREPGE NLYFQGMEER GDSEPTPGCS GLGPGGVRGF GDGGGAPSWA PEDAWMGTH PKYLEMMELD IGDATQVYVA FLVYLDLMES KSWHEVNCVG LPELQLICLV GTEIEGEGLQ TVVPTPITAS L SHNRIREI LKASRKLQGD PDLPMSFTLA IVESDSTIVY YKLTDGFMLP DPQNISLRR

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Macromolecule #5: pre-tRNA Arg TCT 3-2

MacromoleculeName: pre-tRNA Arg TCT 3-2 / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.747045 KDa
SequenceString:
GGCUCUGUGG CGCAAUGGAU AGCGCAUUGG ACUUCUAGAU AGUUAGAGAA AUUCAAAGGU UGUGGGUUCG AGUCCCACCA GAGUCGCCA

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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: 7.4
VitrificationCryogen name: ETHANE
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 63.0 e/Å2

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 282863
FSC plot (resolution estimation)

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