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- PDB-7d0f: cryo-EM structure of a pre-catalytic group II intron RNP -

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Basic information

Entry
Database: PDB / ID: 7d0f
Titlecryo-EM structure of a pre-catalytic group II intron RNP
Components
  • Group II intron-encoded protein LtrA
  • RNA (738-MER)
KeywordsTRANSFERASE/RNA / catalytic RNA / RNA-protein interactions / group II intron / 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: 5 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 10, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 30, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 21, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.year / _citation_author.name
Revision 1.2Nov 18, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-30532
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: RNA (738-MER)
C: Group II intron-encoded protein LtrA


Theoretical massNumber of molelcules
Total (without water)365,7402
Polymers365,7402
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6030 Å2
ΔGint-41 kcal/mol
Surface area115090 Å2

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Components

#1: RNA chain RNA (738-MER)


Mass: 295453.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Gene: LtrB
Production host: Lactococcus lactis subsp. cremoris (lactic acid bacteria)
Strain (production host): IL1403
#2: 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

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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

Component
IDNameTypeEntity IDParent-IDSource
1a group II intron --LtrB complex with its reverse transcriptaseCOMPLEXall0RECOMBINANT
2group II intron-LtrB RNACOMPLEX#11RECOMBINANT
3LtrACOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Lactococcus lactis subsp. cremoris (lactic acid bacteria)1359
33Lactococcus lactis subsp. cremoris (lactic acid bacteria)1359
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Lactococcus lactis subsp. cremoris (lactic acid bacteria)1359IL1403
33Lactococcus lactis subsp. cremoris (lactic acid bacteria)1359IL1403
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285.15 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameCategory
7UCSF Chimeramodel fitting
13PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 203373 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5G2X

5g2x


Accession code: 5G2X / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00721469
ELECTRON MICROSCOPYf_angle_d1.07832655
ELECTRON MICROSCOPYf_dihedral_angle_d24.48811123
ELECTRON MICROSCOPYf_chiral_restr0.0534239
ELECTRON MICROSCOPYf_plane_restr0.0071366

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