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- PDB-7uvt: Kinetically trapped misfolded state of the Tetrahymena ribozyme -

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Basic information

Entry
Database: PDB / ID: 7uvt
TitleKinetically trapped misfolded state of the Tetrahymena ribozyme
ComponentsRNA (386-MER)
KeywordsRNA / ribozyme / catalytic RNA / folding intermediate / misfolded / kinetic trap / M state / Tetrahymena
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBonilla, S.L. / Vicens, Q. / Kieft, J.S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118070 United States
Howard Hughes Medical Institute (HHMI)Hanna H. Gray Postdoctoral Fellowship United States
CitationJournal: Sci Adv / Year: 2022
Title: Cryo-EM reveals an entangled kinetic trap in the folding of a catalytic RNA.
Authors: Steve L Bonilla / Quentin Vicens / Jeffrey S Kieft /
Abstract: Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of these misfolded states, but they are ...Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of these misfolded states, but they are difficult to characterize because of their transient and potentially conformationally dynamic nature. We used cryo-electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the group I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.
History
DepositionMay 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 31, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 7, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA (386-MER)


Theoretical massNumber of molelcules
Total (without water)125,0971
Polymers125,0971
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (386-MER)


Mass: 125096.773 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena thermophila (eukaryote) / References: GenBank: 10832

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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: apo L-21 ScaI Tetrahymena Ribozyme RNA / Type: COMPLEX / Details: Misfolded state. / Entity ID: all / Source: NATURAL
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Buffer solutionpH: 7
Details: Buffer contained 50 mM NaMOPS, pH 7.0 and 10 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 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: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 32 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98071 / Symmetry type: POINT

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