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- EMDB-35694: Cryo-EM structure of wild-type human tRNA Splicing Endonuclease C... -

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Basic information

Entry
Database: EMDB / ID: EMD-35694
TitleCryo-EM structure of wild-type human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG at 3.19 A resolution
Map data
Sample
  • Complex: Human pre-tRNA splicing complex TSEN with substrate RNA
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen15
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen2
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen34
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen54
    • RNA: RNA (88-MER)
  • Ligand: MAGNESIUM ION
KeywordstRNA splicing / TSEN / Cryo-EM / endonuclease / STRUCTURAL PROTEIN
Function / homology
Function and homology information


tRNA-intron endonuclease complex / tRNA-type intron splice site recognition and cleavage / tRNA-intron lyase / tRNA-intron lyase activity / tRNA splicing, via endonucleolytic cleavage and ligation / tRNA processing in the nucleus / tRNA processing / mRNA processing / nucleic acid binding / lyase activity ...tRNA-intron endonuclease complex / tRNA-type intron splice site recognition and cleavage / tRNA-intron lyase / tRNA-intron lyase activity / tRNA splicing, via endonucleolytic cleavage and ligation / tRNA processing in the nucleus / tRNA processing / mRNA processing / nucleic acid binding / lyase activity / centrosome / nucleolus / nucleoplasm / cytosol / cytoplasm
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: 3.19 Å
AuthorsSun Y / Zhang Y / Yuan L / Han Y
Funding support1 items
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, 2023-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateOct 18, 2023-
Current statusOct 18, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35694.png

Downloads & links

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Map

FileDownload / File: emd_35694.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 211. Å		1.06 Å/pix.
x 200 pix.
= 211. Å		1.06 Å/pix.
x 200 pix.
= 211. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.055 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.495
Minimum - Maximum-3.7847893 - 6.157538
Average (Standard dev.)0.002467771 (±0.14946038)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 210.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35694_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35694_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Human pre-tRNA splicing complex TSEN with substrate RNA

EntireName: Human pre-tRNA splicing complex TSEN with substrate RNA
Components
  • Complex: Human pre-tRNA splicing complex TSEN with substrate RNA
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen15
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen2
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen34
    • Protein or peptide: tRNA-splicing endonuclease subunit Sen54
    • RNA: RNA (88-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Human pre-tRNA splicing complex TSEN with substrate RNA

SupramoleculeName: Human pre-tRNA splicing complex TSEN with substrate RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: tRNA-splicing endonuclease subunit Sen15

MacromoleculeName: tRNA-splicing endonuclease subunit Sen15 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 19.760371 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MHHHHHHGSM EERGDSEPTP GCSGLGPGGV RGFGDGGGAP SWAPEDAWMG THPKYLEMME LDIGDATQVY VAFLVYLDLM ESKSWHEVN CVGLPELQLI CLVGTEIEGE GLQTVVPTPI TASLSHNRIR EILKASRKLQ GDPDLPMSFT LAIVESDSTI V YYKLTDGF MLPDPQNISL RR

UniProtKB: tRNA-splicing endonuclease subunit Sen15

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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: 53.326895 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAEAVFHAPK RKRRVYETYE SPLPIPFGQD HGPLKEFKIF RAEMINNNVI VRNAEDIEQL YGKGYFGKGI LSRSRPSFTI SDPKLVAKW KDMKTNMPII TSKRYQHSVE WAAELMRRQG QDESTVRRIL KDYTKPLEHP PVKRNEEAQV HDKLNSGMVS N MEGTAGGE ...String:
MAEAVFHAPK RKRRVYETYE SPLPIPFGQD HGPLKEFKIF RAEMINNNVI VRNAEDIEQL YGKGYFGKGI LSRSRPSFTI SDPKLVAKW KDMKTNMPII TSKRYQHSVE WAAELMRRQG QDESTVRRIL KDYTKPLEHP PVKRNEEAQV HDKLNSGMVS N MEGTAGGE RPSVVNGDSG KSGGVGDPRE PLGCLQEGSG CHPTTESFEK SVREDASPLP HVCCCKQDAL ILQRGLHHED GS QHIGLLH PGDRGPDHEY VLVEEAECAM SEREAAPNEE LVQRNRLICR RNPYRIFEYL QLSLEEAFFL VYALGCLSIY YEK EPLTIV KLWKAFTVVQ PTFRTTYMAY HYFRSKGWVP KVGLKYGTDL LLYRKGPPFY HASYSVIIEL VDDHFEGSLR RPLS WKSLA ALSRVSVNVS KELMLCYLIK PSTMTDKEME SPECMKRIKV QEVILSRWVS SRERSDQDDL

UniProtKB: tRNA-splicing endonuclease subunit Sen2

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Macromolecule #3: tRNA-splicing endonuclease subunit Sen34

MacromoleculeName: tRNA-splicing endonuclease subunit Sen34 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: tRNA-intron lyase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 33.694887 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MLVVEVANGR SLVWGAEAVQ ALRERLGVGG RTVGALPRGP RQNSRLGLPL LLMPEEARLL AEIGAVTLVS APRPDSRHHS LALTSFKRQ QEESFQEQSA LAAEARETRR QELLEKITEG QAAKKQKLEQ ASGASSSQEA GSSQAAKEDE TSDGQASGEQ E EAGPSSSQ ...String:
MLVVEVANGR SLVWGAEAVQ ALRERLGVGG RTVGALPRGP RQNSRLGLPL LLMPEEARLL AEIGAVTLVS APRPDSRHHS LALTSFKRQ QEESFQEQSA LAAEARETRR QELLEKITEG QAAKKQKLEQ ASGASSSQEA GSSQAAKEDE TSDGQASGEQ E EAGPSSSQ AGPSNGVAPL PRSALLVQLA TARPRPVKAR PLDWRVQSKD WPHAGRPAHE LRYSIYRDLW ERGFFLSAAG KF GGDFLVY PGDPLRFHAH YIAQCWAPED TIPLQDLVAA GRLGTSVRKT LLLCSPQPDG KVVYTSLQWA SLQ

UniProtKB: tRNA-splicing endonuclease subunit Sen34

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Macromolecule #4: tRNA-splicing endonuclease subunit Sen54

MacromoleculeName: tRNA-splicing endonuclease subunit Sen54 / type: protein_or_peptide / ID: 4 / Details: GST tag was removed by TEV protease / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 59.451766 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SYWDMEPEPE PAAVEVPAGR VLSARELFAA RSRSQKLPQR SHGPKDFLPD GSAAQAERLR RCREELWQLL AEQRVERLGS LVAAEWRPE EGFVELKSPA GKFWQTMGFS EQGRQRLHPE EALYLLECGS IHLFHQDLPL SIQEAYQLLL TDHTVTFLQY Q VFSHLKRL ...String:
SYWDMEPEPE PAAVEVPAGR VLSARELFAA RSRSQKLPQR SHGPKDFLPD GSAAQAERLR RCREELWQLL AEQRVERLGS LVAAEWRPE EGFVELKSPA GKFWQTMGFS EQGRQRLHPE EALYLLECGS IHLFHQDLPL SIQEAYQLLL TDHTVTFLQY Q VFSHLKRL GYVVRRFQPS SVLSPYERQL NLDASVQHLE DGDGKRKRSS SSPRSINKKA KALDNSLQPK SLAASSPPPC SQ PSQCPEE KPQESSPMKG PGGPFQLLGS LGPSPGPARE GVGCSWESGR AENGVTGAGK RRWNFEQISF PNMASDSRHT LLR APAPEL LPANVAGRET DAESWCQKLN QRKEKLSRRE REHHAEAAQF QEDVNADPEV QRCSSWREYK ELLQRRQVQR SQRR APHLW GQPVTPLLSP GQASSPAVVL QHISVLQTTH LPDGGARLLE KSGGLEIIFD VYQADAVATF RKNNPGKPYA RMCIS GFDE PVPDLCSLKR LSYQSGDVPL IFALVDHGDI SFYSFRDFTL PQDVGH

UniProtKB: tRNA-splicing endonuclease subunit Sen54

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Macromolecule #5: RNA (88-MER)

MacromoleculeName: RNA (88-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.440879 KDa
SequenceString:
GGCUCUGUGG CGCAAUGGAU AGCGCAUUGG ACUUCUAGUG ACGAAUAGAG CAAUUCAAAG GUUGUGGGUU CGAAUCCCAC CAGAGUCG

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
1.0 mMMgCl2Magnesium chloride
5.0 mMDTTDithiothreitol
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsProtein was incubated with RNA on ice for 45 minutes

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 335714
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: predicted
Output model

PDB-8iss:
Cryo-EM structure of wild-type human tRNA Splicing Endonuclease Complex bound to pre-tRNA-ARG at 3.19 A resolution

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