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- PDB-7d1a: cryo-EM structure of a group II intron RNP complexed with its rev... -

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Basic information

Entry
Database: PDB / ID: 7d1a
Titlecryo-EM structure of a group II intron RNP complexed with its reverse transcriptase
Components
  • Group II intron-encoded protein LtrA
  • RNA (5'-R(P*CP*AP*CP*AP*UP*CP*CP*AP*UP*AP*AP*C)-3')
  • RNA (692-MER)
KeywordsTRANSFERASE/RNA / catalytic RNA / RNA-protein interactions / 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
RNA / RNA (> 10) / RNA (> 100) / Group II intron-encoded protein LtrA
Similarity search - Component
Biological speciesLactococcus lactis subsp. cremoris (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLiu, N. / Dong, X.L. / Hu, C.X. / Zeng, J.W. / Wang, J.W. / Wang, J. / Wang, H.W. / Belfort, M.
Funding support China, United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)31825009 China
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM39422; 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 14, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 18, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
C: Group II intron-encoded protein LtrA
A: RNA (692-MER)
B: RNA (5'-R(P*CP*AP*CP*AP*UP*CP*CP*AP*UP*AP*AP*C)-3')


Theoretical massNumber of molelcules
Total (without water)365,7193
Polymers365,7193
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9120 Å2
ΔGint-63 kcal/mol
Surface area119140 Å2

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Components

#1: Protein Group II intron-encoded protein LtrA


Mass: 70286.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Gene: ltrA, matR
Production host: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Strain (production host): IL1403
References: UniProt: P0A3U0, RNA-directed DNA polymerase, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (692-MER)


Mass: 291363.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Production host: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Strain (production host): IL1403
#3: RNA chain RNA (5'-R(P*CP*AP*CP*AP*UP*CP*CP*AP*UP*AP*AP*C)-3')


Mass: 4068.518 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Production host: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Strain (production host): IL1403

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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: cryo-EM structure of a group II intron RNP complexed with its reverse transcriptase
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Source (recombinant)Organism: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Strain: IL1403
Buffer solutionpH: 8
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 FIELDBright-field microscopy
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1150GATAN K2 SUMMIT (4k x 4k)
2150GATAN K2 SUMMIT (4k x 4k)
3150GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 450296 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00720640
ELECTRON MICROSCOPYf_angle_d1.0431361
ELECTRON MICROSCOPYf_dihedral_angle_d21.73710711
ELECTRON MICROSCOPYf_chiral_restr0.0524068
ELECTRON MICROSCOPYf_plane_restr0.0061332

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