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- PDB-7zrz: Structure of the human tRNA splicing endonuclease defines substra... -

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Basic information

Entry
Database: PDB / ID: 7zrz
TitleStructure of the human tRNA splicing endonuclease defines substrate recognition
Components
  • pre-tRNA Arg TCT 3-2
  • tRNA-splicing endonuclease subunit Sen15
  • tRNA-splicing endonuclease subunit Sen2
  • tRNA-splicing endonuclease subunit Sen34
  • tRNA-splicing endonuclease subunit Sen54
KeywordsRNA BINDING PROTEIN / RNP / endonuclease / tRNA / splicing
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
RNA / RNA (> 10) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsSekulovski, S. / Trowitzsch, S.
Funding support Germany, 2items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2023Provider: repository / Type: Initial release
Revision 1.1May 31, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jun 7, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3Jun 28, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AP1: tRNA-splicing endonuclease subunit Sen34
BP4: tRNA-splicing endonuclease subunit Sen2
CP1: tRNA-splicing endonuclease subunit Sen54
DP1: tRNA-splicing endonuclease subunit Sen15
ZN1: pre-tRNA Arg TCT 3-2


Theoretical massNumber of molelcules
Total (without water)144,1315
Polymers144,1315
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16820 Å2
ΔGint-97 kcal/mol
Surface area41200 Å2
MethodPISA

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Components

#1: Protein tRNA-splicing endonuclease subunit Sen34 / Leukocyte receptor cluster member 5 / tRNA-intron endonuclease Sen34 / HsSen34


Mass: 28805.811 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN34, LENG5, SEN34 / Production host: Homo sapiens (human) / References: UniProt: Q9BSV6, tRNA-intron lyase
#2: Protein tRNA-splicing endonuclease subunit Sen2 / tRNA-intron endonuclease Sen2 / HsSen2


Mass: 30165.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN2, SEN2 / Production host: Homo sapiens (human) / References: UniProt: Q8NCE0, tRNA-intron lyase
#3: Protein tRNA-splicing endonuclease subunit Sen54 / SEN54 homolog / HsSEN54 / tRNA-intron endonuclease Sen54


Mass: 32551.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN54, SEN54 / Production host: Homo sapiens (human) / References: UniProt: Q7Z6J9
#4: Protein tRNA-splicing endonuclease subunit Sen15 / SEN15 homolog / HsSEN15 / tRNA-intron endonuclease Sen15


Mass: 23860.889 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN15, C1orf19, SEN15 / Production host: Homo sapiens (human) / References: UniProt: Q8WW01
#5: RNA chain pre-tRNA Arg TCT 3-2


Mass: 28747.045 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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: Human TSEN with pre-tRNA-Arg-TCT / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 63 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 282863 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 25.35 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00226903
ELECTRON MICROSCOPYf_angle_d0.50589737
ELECTRON MICROSCOPYf_chiral_restr0.03731135
ELECTRON MICROSCOPYf_plane_restr0.004958
ELECTRON MICROSCOPYf_dihedral_angle_d12.87611601

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