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- PDB-8hmy: Cryo-EM structure of the human pre-catalytic TSEN/pre-tRNA complex -

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Basic information

Entry
Database: PDB / ID: 8hmy
TitleCryo-EM structure of the human pre-catalytic TSEN/pre-tRNA complex
Components
  • (tRNA-splicing endonuclease subunit ...) x 3
  • Chromosome 1 open reading frame 19, isoform CRA_a
  • Polyribonucleotide 5'-hydroxyl-kinase Clp1
  • Pre-tRNA
KeywordsRNA BINDING PROTEIN/RNA / pre-tRNA splicing / TSEN / pre-tRNA / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


tRNA-intron endonuclease complex / ATP-dependent polyribonucleotide 5'-hydroxyl-kinase activity / tRNA-type intron splice site recognition and cleavage / polynucleotide 5'-hydroxyl-kinase / tRNA-intron lyase / tRNA-intron endonuclease activity / ATP-dependent polydeoxyribonucleotide 5'-hydroxyl-kinase activity / polynucleotide 5'-hydroxyl-kinase activity / mRNA cleavage factor complex / tRNA splicing, via endonucleolytic cleavage and ligation ...tRNA-intron endonuclease complex / ATP-dependent polyribonucleotide 5'-hydroxyl-kinase activity / tRNA-type intron splice site recognition and cleavage / polynucleotide 5'-hydroxyl-kinase / tRNA-intron lyase / tRNA-intron endonuclease activity / ATP-dependent polydeoxyribonucleotide 5'-hydroxyl-kinase activity / polynucleotide 5'-hydroxyl-kinase activity / mRNA cleavage factor complex / tRNA splicing, via endonucleolytic cleavage and ligation / global gene silencing by mRNA cleavage / cerebellar cortex development / Processing of Intronless Pre-mRNAs / RISC complex assembly / mRNA 3'-end processing / mRNA 3'-end processing / tRNA processing in the nucleus / RNA Polymerase II Transcription Termination / Processing of Capped Intron-Containing Pre-mRNA / mRNA processing / nucleic acid binding / lyase activity / centrosome / nucleolus / nucleoplasm / ATP binding / nucleus / 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 / Pre-mRNA cleavage complex subunit Clp1, C-terminal / Polyribonucleotide 5-hydroxyl-kinase Clp1 / Clp1, N-terminal beta-sandwich domain ...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 / Pre-mRNA cleavage complex subunit Clp1, C-terminal / Polyribonucleotide 5-hydroxyl-kinase Clp1 / Clp1, N-terminal beta-sandwich domain / Clp1, C-terminal domain superfamily / Clp1, N-terminal beta-sandwich domain superfamily / Pre-mRNA cleavage complex II protein Clp1 / N-terminal beta-sandwich domain of polyadenylation factor / 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 / Polyribonucleotide 5'-hydroxyl-kinase Clp1, P-loop domain / Polyribonucleotide 5-hydroxyl-kinase Clp1/Grc3 / mRNA cleavage and polyadenylation factor CLP1 P-loop / tRNA endonuclease-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Chromosome 1 open reading frame 19, isoform CRA_a / tRNA-splicing endonuclease subunit Sen54 / tRNA-splicing endonuclease subunit Sen2 / Polyribonucleotide 5'-hydroxyl-kinase Clp1 / tRNA-splicing endonuclease subunit Sen34
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsZhang, X. / Yang, F. / Zhan, X. / Shi, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of pre-tRNA intron removal by human tRNA splicing endonuclease.
Authors: Xiaofeng Zhang / Fenghua Yang / Xiechao Zhan / Tong Bian / Zhihan Xing / Yichen Lu / Yigong Shi /
Abstract: Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease (TSEN) comprising four subunits: ...Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease (TSEN) comprising four subunits: TSEN2, TSEN15, TSEN34, and TSEN54. Here, we report the cryo-EM structures of human TSEN bound to full-length pre-tRNA in the pre-catalytic and post-catalytic states at average resolutions of 2.94 and 2.88 Å, respectively. Human TSEN features an extended surface groove that holds the L-shaped pre-tRNA. The mature domain of pre-tRNA is recognized by conserved structural elements of TSEN34, TSEN54, and TSEN2. Such recognition orients the anticodon stem of pre-tRNA and places the 3'-splice site and 5'-splice site into the catalytic centers of TSEN34 and TSEN2, respectively. The bulk of the intron sequences makes no direct interaction with TSEN, explaining why pre-tRNAs of varying introns can be accommodated and cleaved. Our structures reveal the molecular ruler mechanism of pre-tRNA cleavage by TSEN.
History
DepositionDec 6, 2022Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1May 3, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2May 29, 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 Sen2
B: tRNA-splicing endonuclease subunit Sen34
T: Pre-tRNA
C: tRNA-splicing endonuclease subunit Sen54
D: Chromosome 1 open reading frame 19, isoform CRA_a
E: Polyribonucleotide 5'-hydroxyl-kinase Clp1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,76312
Polymers261,6176
Non-polymers1466
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 ... , 3 types, 3 molecules ABC

#1: Protein tRNA-splicing endonuclease subunit Sen2 / tRNA-intron endonuclease Sen2 / HsSen2


Mass: 55391.945 Da / Num. of mol.: 1 / Mutation: H377A
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
#2: Protein tRNA-splicing endonuclease subunit Sen34 / Leukocyte receptor cluster member 5 / tRNA-intron endonuclease Sen34 / HsSen34


Mass: 35759.938 Da / Num. of mol.: 1 / Mutation: H255A
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
#4: Protein tRNA-splicing endonuclease subunit Sen54 / SEN54 homolog / HsSEN54 / tRNA-intron endonuclease Sen54


Mass: 61032.340 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

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Protein , 2 types, 2 molecules DE

#5: Protein Chromosome 1 open reading frame 19, isoform CRA_a / tRNA-splicing endonuclease subunit Sen15


Mass: 22796.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN15, C1orf19, hCG_40901 / Production host: Homo sapiens (human) / References: UniProt: A0A2U3TZM3
#6: Protein Polyribonucleotide 5'-hydroxyl-kinase Clp1 / Polyadenylation factor Clp1 / Polynucleotide kinase Clp1 / Pre-mRNA cleavage complex II protein Clp1


Mass: 49837.504 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Author stated that the EM density map for the chain E is relatively weak and could be assigned in the low resolution map.
Source: (gene. exp.) Homo sapiens (human) / Gene: CLP1, HEAB / Production host: Homo sapiens (human)
References: UniProt: Q92989, polynucleotide 5'-hydroxyl-kinase

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RNA chain / Non-polymers , 2 types, 7 molecules T

#3: RNA chain Pre-tRNA


Mass: 36798.828 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / 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: The human pre-catalytic TSEN/pre-tRNA complex / Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 199001 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410202
ELECTRON MICROSCOPYf_angle_d0.59914244
ELECTRON MICROSCOPYf_dihedral_angle_d14.3852166
ELECTRON MICROSCOPYf_chiral_restr0.041649
ELECTRON MICROSCOPYf_plane_restr0.0051496

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