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- PDB-7ych: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoi... -

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Basic information

Entry
Database: PDB / ID: 7ych
TitleCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the first-step self-splicing
Components
  • RNA (393-MER)
  • RNA (5'-R(*CP*CP*CP*UP*CP*UP*AP*AP*AP*CP*C)-3')
KeywordsRNA / Tetrahymena ribozyme / first step of self-splicing / conformation 3
Function / homologyGUANOSINE-5'-TRIPHOSPHATE / : / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsZhang, X. / Li, S. / Pintilie, G. / Palo, M.Z. / Zhang, K.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Snapshots of the first-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM.
Authors: Xiaojing Zhang / Shanshan Li / Grigore Pintilie / Michael Z Palo / Kaiming Zhang /
Abstract: Tetrahymena ribozyme is a group I intron, whose self-splicing is the result of two sequential ester-transfer reactions. To understand how it facilitates catalysis in the first self-splicing reaction, ...Tetrahymena ribozyme is a group I intron, whose self-splicing is the result of two sequential ester-transfer reactions. To understand how it facilitates catalysis in the first self-splicing reaction, we used cryogenic electron microscopy (cryo-EM) to resolve the structures of L-16 Tetrahymena ribozyme complexed with a 11-nucleotide 5'-splice site analog substrate. Four conformations were achieved to 4.14, 3.18, 3.09 and 2.98 Å resolutions, respectively, corresponding to different splicing intermediates during the first enzymatic reaction. Comparison of these structures reveals structural alterations, including large conformational changes in IGS/IGSext (P1-P1ext duplex) and J5/4, as well as subtle local rearrangements in the G-binding site. These structural changes are required for the enzymatic activity of the Tetrahymena ribozyme. Our study demonstrates the ability of cryo-EM to capture dynamic RNA structural changes, ushering in a new era in the analysis of RNA structure-function by cryo-EM.
History
DepositionJul 1, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 19, 2023Group: Database references / Structure summary / Category: audit_author / citation_author / Item: _audit_author.name / _citation_author.name
Revision 1.2Aug 16, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
B: RNA (5'-R(*CP*CP*CP*UP*CP*UP*AP*AP*AP*CP*C)-3')
N: RNA (393-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,9946
Polymers130,3982
Non-polymers5964
Water00
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 (5'-R(*CP*CP*CP*UP*CP*UP*AP*AP*AP*CP*C)-3')


Mass: 3386.082 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena (eukaryote)
#2: RNA chain RNA (393-MER)


Mass: 127011.883 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena (eukaryote)
Production host: in vitro transcription vector pT7-Fluc(deltai) (others)
References: GenBank: 10832
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 Tetrahymena ribozyme conformation 3 undergoing the first-step self-splicing
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: YES
Source (natural)Organism: Tetrahymena (eukaryote)
Source (recombinant)Organism: in vitro transcription vector pT7-Fluc(deltai) (others)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding 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 FIELD / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
12cryoSPARC2.3classification
13cryoSPARC2.33D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3668247
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202401 / Symmetry type: POINT

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