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- PDB-6wls: Tetrahymena ribozyme models, 6.8 Angstrom resolution -

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Entry
Database: PDB / ID: 6wls
TitleTetrahymena ribozyme models, 6.8 Angstrom resolution
ComponentsRNA (388-MER)
KeywordsRNA / ribozyme
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsKappel, K. / Zhang, K. / Su, Z. / Watkins, A.M. / Kladwang, W. / Li, S. / Pintilie, G. / Topkar, V.V. / Rangan, R. / Zheludev, I.N. ...Kappel, K. / Zhang, K. / Su, Z. / Watkins, A.M. / Kladwang, W. / Li, S. / Pintilie, G. / Topkar, V.V. / Rangan, R. / Zheludev, I.N. / Yesselman, J.D. / Chiu, W. / Das, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorS10 OD021600 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21 AI145647 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832, R01GM079429, U54GM103297, R35 GM112579 United States
CitationJournal: Nat Methods / Year: 2020
Title: Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures.
Authors: Kalli Kappel / Kaiming Zhang / Zhaoming Su / Andrew M Watkins / Wipapat Kladwang / Shanshan Li / Grigore Pintilie / Ved V Topkar / Ramya Rangan / Ivan N Zheludev / Joseph D Yesselman / Wah Chiu / Rhiju Das /
Abstract: The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve ...The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
History
DepositionApr 20, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 15, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 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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Assembly

Deposited unit
A: RNA (388-MER)


Theoretical massNumber of molelcules
Total (without water)125,4031
Polymers125,4031
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area61880 Å2
Number of models20

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Components

#1: RNA chain RNA (388-MER)


Mass: 125402.945 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: Tetrahymena ribozyme / Type: COMPLEX / Details: RNA generated by in vitro transcription / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.126 MDa / Experimental value: YES
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74621 / Symmetry type: POINT

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