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- PDB-7uxa: Human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG -

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Basic information

Entry
Database: PDB / ID: 7uxa
TitleHuman tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG
Components
  • (tRNA-splicing endonuclease subunit ...) x 4
  • RNA (78-MER)
KeywordsSPLICING/RNA / splicing 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
: / 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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsStanley, R.E. / Hayne, C.K.
Funding support United States, 8items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1May 31, 2023Group: Database references / Category: citation
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
Revision 1.3Jul 5, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: tRNA-splicing endonuclease subunit Sen34
B: tRNA-splicing endonuclease subunit Sen15
C: tRNA-splicing endonuclease subunit Sen2
D: tRNA-splicing endonuclease subunit Sen54
E: RNA (78-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,6447
Polymers203,5955
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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TRNA-splicing endonuclease subunit ... , 4 types, 4 molecules ABCD

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


Mass: 37941.316 Da / Num. of mol.: 1 / Mutation: Y247A,H255A,K286A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN34, LENG5, SEN34 / Cell line (production host): HEK293 freestyle / Production host: Homo sapiens (human) / References: UniProt: Q9BSV6, tRNA-intron lyase
#2: Protein tRNA-splicing endonuclease subunit Sen15 / SEN15 homolog / HsSEN15 / tRNA-intron endonuclease Sen15


Mass: 19996.619 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN15, C1orf19, SEN15 / Cell line (production host): HEK293 Freestyle / Production host: Homo sapiens (human) / References: UniProt: Q8WW01
#3: Protein tRNA-splicing endonuclease subunit Sen2 / tRNA-intron endonuclease Sen2 / HsSen2


Mass: 55352.961 Da / Num. of mol.: 1 / Mutation: Y369A, H377A, K416A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN2, SEN2 / Cell line (production host): HEK293 Freestyle / Production host: Homo sapiens (human) / References: UniProt: Q8NCE0, tRNA-intron lyase
#4: Protein tRNA-splicing endonuclease subunit Sen54 / SEN54 homolog / HsSEN54 / tRNA-intron endonuclease Sen54


Mass: 61863.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN54, SEN54 / Cell line (production host): HEK293 Freestyle / Production host: Homo sapiens (human) / References: UniProt: Q7Z6J9

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RNA chain / Non-polymers , 2 types, 3 molecules E

#5: RNA chain RNA (78-MER)


Mass: 28440.879 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: GenBank: 473010
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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 tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: .203 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31synthetic construct (others)32630
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 Freestyle
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company /
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Mode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Specimen-ID: 1

IDAccelerating voltage (kV)ModelNominal magnification (X)
1200FEI TALOS ARCTICA45000
2300FEI TITAN KRIOS81000
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1154GATAN K2 SUMMIT (4k x 4k)
2260GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryImaging-ID
4cryoSPARC3.2.0CTF correction
15cryoSPARC3.2.03D reconstruction
30SerialEMimage acquisition2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 570104
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152031 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 77.39 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00328469
ELECTRON MICROSCOPYf_angle_d0.511869
ELECTRON MICROSCOPYf_chiral_restr0.03571384
ELECTRON MICROSCOPYf_plane_restr0.00391229
ELECTRON MICROSCOPYf_dihedral_angle_d11.9453360

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