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- PDB-7r6m: Post-2S intermediate of the Tetrahymena group I intron, symmetry-... -

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Basic information

Entry
Database: PDB / ID: 7r6m
TitlePost-2S intermediate of the Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
Components
  • Group I intronGroup I catalytic intron
  • Ligated exon mimic of the Group I intron
KeywordsRNA / Ribozyme / Group I intron / catalytic RNA
Function / homology: / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsThelot, F. / Liu, D. / Liao, M. / Yin, P.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM122797 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5DP1GM133052 United States
National Science Foundation (NSF, United States)CMMI-1333215 United States
National Science Foundation (NSF, United States)CMMI-1344915 United States
National Science Foundation (NSF, United States)CBET-1729397 United States
CitationJournal: Nat Methods / Year: 2022
Title: Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly.
Authors: Di Liu / François A Thélot / Joseph A Piccirilli / Maofu Liao / Peng Yin /
Abstract: High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural ...High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy-ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops)-involves installing kissing-loop sequences onto the functionally nonessential stems of RNAs for homomeric self-assembly into closed rings with multiplied molecular weights and mitigated structural flexibility. ROCK enables cryo-EM reconstruction of the Tetrahymena group I intron at 2.98-Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA, including the previously unknown peripheral domains. ROCK is further applied to two smaller RNAs-the Azoarcus group I intron and the FMN riboswitch, revealing the conformational change of the former and the bound ligand in the latter. ROCK holds promise to greatly facilitate the use of cryo-EM in RNA structural studies.
History
DepositionJun 22, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 4, 2022Provider: repository / Type: Initial release
Revision 1.1May 18, 2022Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 1, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Group I intron
B: Ligated exon mimic of the Group I intron
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,68415
Polymers132,3682
Non-polymers31613
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, native gel supports the dimeric assembly, the submitted map and model are for the symmetry-expanded monomer
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2420 Å2
ΔGint-119 kcal/mol
Surface area54720 Å2

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Components

#1: RNA chain Group I intron / Group I catalytic intron


Mass: 128011.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena thermophila (eukaryote) / References: GenBank: V01416
#2: DNA/RNA hybrid Ligated exon mimic of the Group I intron


Mass: 4356.657 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena thermophila (eukaryote)
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 13 / 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: Post-2S intermediate of Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
Type: COMPLEX / Entity ID: #1-#2 / Source: SYNTHETIC
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-acetateTris-acetate1
230 mMMagnesium chlorideMgCl21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: OTHER / Nominal magnification: 105000 X / Cs: 2.7 mm
Image recordingElectron dose: 47 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113548 / Symmetry type: POINT

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