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- EMDB-30533: Cryo-EM structure of a pre-catalytic group II intron -

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Basic information

Entry
Database: EMDB / ID: EMD-30533
TitleCryo-EM structure of a pre-catalytic group II intron
Map data
Sample
  • Complex: a group II intron -- LtrB complex with its reverse transcriptase
    • Complex: RNA
      • RNA: RNA (714-MER)
    • Complex: LtrB
      • Protein or peptide: Group II intron-encoded protein LtrA
Keywordsgroup II intron / RNA-protein complex / pre-catalytic / TRANSFERASE-RNA complex
Function / homology
Function and homology information


intron homing / mRNA processing / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / endonuclease activity / Hydrolases; Acting on ester bonds
Similarity search - Function
Domain X / : / Type II intron maturase / AI2M/AI1M-like, HNH endonuclease / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Group II intron-encoded protein LtrA
Similarity search - Component
Biological speciesLactococcus lactis subsp. cremoris (lactic acid bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsLiu N / Dong XL
Funding support China, United States, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)31825009 China
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM39422 and GM44844 United States
CitationJournal: Nucleic Acids Res / Year: 2020
Title: Exon and protein positioning in a pre-catalytic group II intron RNP primed for splicing.
Authors: Nan Liu / Xiaolong Dong / Cuixia Hu / Jianwei Zeng / Jiawei Wang / Jia Wang / Hong-Wei Wang / Marlene Belfort /
Abstract: Group II introns are the putative progenitors of nuclear spliceosomal introns and use the same two-step splicing pathway. In the cell, the intron RNA forms a ribonucleoprotein (RNP) complex with the ...Group II introns are the putative progenitors of nuclear spliceosomal introns and use the same two-step splicing pathway. In the cell, the intron RNA forms a ribonucleoprotein (RNP) complex with the intron-encoded protein (IEP), which is essential for splicing. Although structures of spliced group II intron RNAs and RNP complexes have been characterized, structural insights into the splicing process remain enigmatic due to lack of pre-catalytic structural models. Here, we report two cryo-EM structures of endogenously produced group II intron RNPs trapped in their pre-catalytic state. Comparison of the catalytically activated precursor RNP to its previously reported spliced counterpart allowed identification of key structural rearrangements accompanying splicing, including a remodeled active site and engagement of the exons. Importantly, altered RNA-protein interactions were observed upon splicing among the RNP complexes. Furthermore, analysis of the catalytically inert precursor RNP demonstrated the structural impact of the formation of the active site on RNP architecture. Taken together, our results not only fill a gap in understanding the structural basis of IEP-assisted group II intron splicing, but also provide parallels to evolutionarily related spliceosomal splicing.
History
DepositionSep 10, 2020-
Header (metadata) releaseSep 30, 2020-
Map releaseSep 30, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7d0g
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30533.png

Downloads & links

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Map

FileDownload / File: emd_30533.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.30654 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.015
Minimum - Maximum-0.017754426 - 0.060868833
Average (Standard dev.)0.00053444365 (±0.0032688896)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions184184184
Spacing184184184
CellA=B=C: 240.40337 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.30653804347831.30653804347831.3065380434783
M x/y/z184184184
origin x/y/z0.0000.0000.000
length x/y/z240.403240.403240.403
α/β/γ90.00090.00090.000
start NX/NY/NZ212211153
NX/NY/NZ80102186
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS184184184
D min/max/mean-0.0180.0610.001

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Supplemental data

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Sample components

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Entire : a group II intron -- LtrB complex with its reverse transcriptase

EntireName: a group II intron -- LtrB complex with its reverse transcriptase
Components
  • Complex: a group II intron -- LtrB complex with its reverse transcriptase
    • Complex: RNA
      • RNA: RNA (714-MER)
    • Complex: LtrB
      • Protein or peptide: Group II intron-encoded protein LtrA

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Supramolecule #1: a group II intron -- LtrB complex with its reverse transcriptase

SupramoleculeName: a group II intron -- LtrB complex with its reverse transcriptase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: RNA

SupramoleculeName: RNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Lactococcus lactis subsp. cremoris (lactic acid bacteria)

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Supramolecule #3: LtrB

SupramoleculeName: LtrB / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lactococcus lactis subsp. cremoris (lactic acid bacteria)

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Macromolecule #1: RNA (714-MER)

MacromoleculeName: RNA (714-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Molecular weightTheoretical: 295.767594 KDa
SequenceString: CACAUCCAUA ACGUGCGCCC AGAUAGGGUG UUAAGUCAAG UAGUUUAAGG UACUACUCUG UAAGAUAACA CAGAAAACAG CCAACCUAA CCGAAAAGCG AAAGCUGAUA CGGGAACAGA GCACGGUUGG AAAGCGAUGA GUUACCUAAA GACAAUCGGG U ACGACUGA ...String:
CACAUCCAUA ACGUGCGCCC AGAUAGGGUG UUAAGUCAAG UAGUUUAAGG UACUACUCUG UAAGAUAACA CAGAAAACAG CCAACCUAA CCGAAAAGCG AAAGCUGAUA CGGGAACAGA GCACGGUUGG AAAGCGAUGA GUUACCUAAA GACAAUCGGG U ACGACUGA GUCGCAAUGU UAAUCAGAUA UAAGGUAUAA GUUGUGUUUA CUGAACGCAA GUUUCUAAUU UCGGUUAUGU GU CGAUAGA GGAAAGUGUC UGAAACCUCU AGUACAAAGA AAGGUAAGUU AUGGUUGUGG ACUUAUCUGU UAUCACCACA UUU GUACAA UCUGUAGGAG AACCUAUGGG AACGAAACGA AAGCGAUGCC GAGAAUCUGA AUUUACCAAG ACUUAACACU AACU GGGGA UACCCUAAAC AAGAAUGCCU AAUAGAAAGG AGGAAAAAGG CUAUAGCACU AGAGCUUGAA AAUCUUGCAA GGGUA CGGA GUACUCGUAG UAGUCUGAGA AGGGUAACGC CCUUUACAUG GCAAAGGGGU ACAGUUAUUG UGUACUAAAA UUAAAA AUU GAUUAGGGAG GAAAACCUCA AAAUGAAACC AACAAUGGCA AUUUUAGAAA GAAUCAGUAA AAAUUCACAA GAAAAUA UA GACGAAGUUU UUACAAGACU UUAUCGUUAU CUUUUACGUC CAGAUAUUUA UUACGUGGCG ACGCGUUGGG AAAUGGCA A UGAUAGCGAA ACAACGUAAA ACUCUUGUUG UAUGCUUUCA UUGUCAUCGU CACGUGAUUC AUAAACACAA GUGAAUUUU UACGAACGAA CAAUAACAGA AUCGUAUACU CCGAGAGGGG UACGUACGGU UCCCGAAGAG GGUGGUGCAA ACCAGUCACA GUAAUGUGA ACAAGGCGGU ACCUCCCUAC UUCACCA

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Macromolecule #2: Group II intron-encoded protein LtrA

MacromoleculeName: Group II intron-encoded protein LtrA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Molecular weightTheoretical: 70.286664 KDa
Recombinant expressionOrganism: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
SequenceString: MKPTMAILER ISKNSQENID EVFTRLYRYL LRPDIYYVAY QNLYSNKGAS TKGILDDTAD GFSEEKIKKI IQSLKDGTYY PQPVRRMYI AKKNSKKMRP LGIPTFTDKL IQEAVRIILE SIYEPVFEDV SHGFRPQRSC HTALKTIKRE FGGARWFVEG D IKGCFDNI ...String:
MKPTMAILER ISKNSQENID EVFTRLYRYL LRPDIYYVAY QNLYSNKGAS TKGILDDTAD GFSEEKIKKI IQSLKDGTYY PQPVRRMYI AKKNSKKMRP LGIPTFTDKL IQEAVRIILE SIYEPVFEDV SHGFRPQRSC HTALKTIKRE FGGARWFVEG D IKGCFDNI DHVTLIGLIN LKIKDMKMSQ LIYKFLKAGY LENWQYHKTY SGTPQGGILS PLLANIYLHE LDKFVLQLKM KF DRESPER ITPEYRELHN EIKRISHRLK KLEGEEKAKV LLEYQEKRKR LPTLPCTSQT NKVLKYVRYA DDFIISVKGS KED CQWIKE QLKLFIHNKL KMELSEEKTL ITHSSQPARF LGYDIRVRRS GTIKRSGKVK KRTLNGSVEL LIPLQDKIRQ FIFD KKIAI QKKDSSWFPV HRKYLIRSTD LEIITIYNSE LRGICNYYGL ASNFNQLNYF AYLMEYSCLK TIASKHKGTL SKTIS MFKD GSGSWGIPYE IKQGKQRRYF ANFSECKSPY QFTDEISQAP VLYGYARNTL ENRLKAKCCE LCGTSDENTS YEIHHV NKV KNLKGKEKWE MAMIAKQRKT LVVCFHCHRH VIHKHK

UniProtKB: Group II intron-encoded protein LtrA

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 391788

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