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- PDB-8iss: Cryo-EM structure of wild-type human tRNA Splicing Endonuclease C... -

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Basic information

Entry
Database: PDB / ID: 8iss
TitleCryo-EM structure of wild-type human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG at 3.19 A resolution
Components
  • (tRNA-splicing endonuclease subunit ...) x 4
  • RNA (88-MER)
KeywordsSTRUCTURAL PROTEIN / tRNA splicing / TSEN / Cryo-EM / endonuclease
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: 3.19 Å
AuthorsSun, Y. / Zhang, Y. / Yuan, L. / Han, Y.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2023
Title: Recognition and cleavage mechanism of intron-containing pre-tRNA by human TSEN endonuclease complex.
Authors: Ling Yuan / Yaoyao Han / Jiazheng Zhao / Yixiao Zhang / Yadong Sun /
Abstract: Removal of introns from transfer RNA precursors (pre-tRNAs) occurs in all living organisms. This is a vital phase in the maturation and functionality of tRNA. Here we present a 3.2 Å-resolution cryo- ...Removal of introns from transfer RNA precursors (pre-tRNAs) occurs in all living organisms. This is a vital phase in the maturation and functionality of tRNA. Here we present a 3.2 Å-resolution cryo-EM structure of an active human tRNA splicing endonuclease complex bound to an intron-containing pre-tRNA. TSEN54, along with the unique regions of TSEN34 and TSEN2, cooperatively recognizes the mature body of pre-tRNA and guides the anticodon-intron stem to the correct position for splicing. We capture the moment when the endonucleases are poised for cleavage, illuminating the molecular mechanism for both 3' and 5' cleavage reactions. Two insertion loops from TSEN54 and TSEN2 cover the 3' and 5' splice sites, respectively, trapping the scissile phosphate in the center of the catalytic triad of residues. Our findings reveal the molecular mechanism for eukaryotic pre-tRNA recognition and cleavage, as well as the evolutionary relationship between archaeal and eukaryotic TSENs.
History
DepositionMar 21, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: tRNA-splicing endonuclease subunit Sen15
B: tRNA-splicing endonuclease subunit Sen2
C: tRNA-splicing endonuclease subunit Sen34
D: tRNA-splicing endonuclease subunit Sen54
E: RNA (88-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,6996
Polymers194,6755
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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 Sen15 / tRNA splicing endonuclease subunit 15 / SEN15 homolog / HsSEN15 / tRNA-intron endonuclease Sen15


Mass: 19760.371 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q8WW01
#2: Protein tRNA-splicing endonuclease subunit Sen2 / tRNA splicing endonuclease subunit 2 / tRNA-intron endonuclease Sen2 / HsSen2


Mass: 53326.895 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN2, SEN2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q8NCE0, tRNA-intron lyase
#3: Protein tRNA-splicing endonuclease subunit Sen34 / tRNA splicing endonuclease subunit 34 / Leukocyte receptor cluster member 5 / tRNA-intron ...tRNA splicing endonuclease subunit 34 / Leukocyte receptor cluster member 5 / tRNA-intron endonuclease Sen34 / HsSen34


Mass: 33694.887 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BSV6, tRNA-intron lyase
#4: Protein tRNA-splicing endonuclease subunit Sen54 / tRNA splicing endonuclease subunit 54 / SEN54 homolog / HsSEN54 / tRNA-intron endonuclease Sen54


Mass: 59451.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GST tag was removed by TEV protease / Source: (gene. exp.) Homo sapiens (human) / Gene: TSEN54, SEN54 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q7Z6J9

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

#5: RNA chain RNA (88-MER)


Mass: 28440.879 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / 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 pre-tRNA splicing complex TSEN with substrate RNA
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
31 mMMagnesium chlorideMgCl21
45 mMDithiothreitolDTT1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Protein was incubated with RNA on ice for 45 minutes
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 49.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 335714 / Symmetry type: POINT
Atomic model buildingDetails: predicted / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059189
ELECTRON MICROSCOPYf_angle_d0.59312832
ELECTRON MICROSCOPYf_dihedral_angle_d13.2091935
ELECTRON MICROSCOPYf_chiral_restr0.0371479
ELECTRON MICROSCOPYf_plane_restr0.0051347

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