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- PDB-7xsl: Misfolded Tetrahymena ribozyme conformation 2 -

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Entry
Database: PDB / ID: 7xsl
TitleMisfolded Tetrahymena ribozyme conformation 2
ComponentsRNA (388-MER)
KeywordsRNA / Misfolded Tetrahymena Ribozyme / Topological Crossing / Cryo-EM / refolding
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsLi, S. / Palo, M. / Pintilie, G. / Zhang, X. / Su, Z. / Kappel, K. / Chiu, W. / Zhang, K. / Das, R.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129541 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122579 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R21 AI145647 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Topological crossing in the misfolded ribozyme resolved by cryo-EM.
Authors: Shanshan Li / Michael Z Palo / Grigore Pintilie / Xiaojing Zhang / Zhaoming Su / Kalli Kappel / Wah Chiu / Kaiming Zhang / Rhiju Das /
Abstract: The group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M) , which has been known to form extensive ...The group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M) , which has been known to form extensive native-like secondary and tertiary structures but is separated by an unknown kinetic barrier from the native state (N). Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the L-21 ScaI ribozyme. Maps of three M substates (M1, M2, M3) and one N state were achieved from a single specimen with overall resolutions of 3.5 Å, 3.8 Å, 4.0 Å, and 3.0 Å, respectively. Comparisons of the structures reveal that all the M substates are highly similar to N, except for rotation of a core helix P7 that harbors the ribozyme's guanosine binding site and the crossing of the strands J7/3 and J8/7 that connect P7 to the other elements in the ribozyme core. This topological difference between the M substates and N state explains the failure of 5'-splice site substrate docking in M, supports a topological isomer model for the slow refolding of M to N due to a trapped strand crossing, and suggests pathways for M-to-N refolding.
History
DepositionMay 14, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 3, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
N: RNA (388-MER)


Theoretical massNumber of molelcules
Total (without water)125,4031
Polymers125,4031
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (388-MER)


Mass: 125402.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena thermophila (eukaryote)
Production host: in vitro transcription vector pT7-TP(deltai) (others)
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: Misfolded Tetrahymena ribozyme conformation 2 / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
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 / Nominal defocus max: 2800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 51.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 42382

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Processing

EM software
IDNameVersionCategory
1EMAN22.3particle selection
2EPU2.7image acquisition
4CTFFIND4CTF correction
11cryoSPARC2.3final Euler assignment
12cryoSPARC2.3classification
13cryoSPARC2.33D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 10414332
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 431156 / Symmetry type: POINT

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