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- EMDB-24281: 5 prime exon-free pre-2S intermediate of the Tetrahymena group I ... -

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Basic information

Entry
Database: EMDB / ID: EMD-24281
Title5 prime exon-free pre-2S intermediate of the Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
Map data5 prime exon-free pre-2S intermediate of Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
Sample
  • Complex: 5 prime exon-free pre-2S intermediate of Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
    • RNA: Group I intron, 5 prime fragmentGroup I catalytic intron
    • Other: Group I intron, 3 prime fragment plus 3 prime exonGroup I catalytic intron
  • Ligand: MAGNESIUM ION
KeywordsRibozyme / Group I intron / catalytic RNA / RNA
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsThelot F / Liu D
Funding support United States, 5 items
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, 2021-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24281.png

Downloads & links

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Map

FileDownload / File: emd_24281.map.gz / Format: CCP4 / Size: 7.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation5 prime exon-free pre-2S intermediate of Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.393
Minimum - Maximum-3.3789575 - 5.539524
Average (Standard dev.)0.000000000010373 (±0.31723043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin15411582
Dimensions137115132
Spacing132137115
CellA: 108.9 Å / B: 113.025 Å / C: 94.875 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : 5 prime exon-free pre-2S intermediate of Tetrahymena group I intr...

EntireName: 5 prime exon-free pre-2S intermediate of Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
Components
  • Complex: 5 prime exon-free pre-2S intermediate of Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
    • RNA: Group I intron, 5 prime fragmentGroup I catalytic intron
    • Other: Group I intron, 3 prime fragment plus 3 prime exonGroup I catalytic intron
  • Ligand: MAGNESIUM ION

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Supramolecule #1: 5 prime exon-free pre-2S intermediate of Tetrahymena group I intr...

SupramoleculeName: 5 prime exon-free pre-2S intermediate of Tetrahymena group I intron, symmetry-expanded monomer from a synthetic dimeric construct
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Synthetically produced: Yes
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Group I intron, 5 prime fragment

MacromoleculeName: Group I intron, 5 prime fragment / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 121.342711 KDa
SequenceString: GGCCGGGUGG AGGGAAAAGU UAUCAGGCAU GCACCUGGUA GCUAGUCUUU AAACCAAUAG AUUGCAUCGG UUUAAAAGGC AAGACCGUC AAAUUGCGGG AAAGGGGUCA ACAGCCGUUC AGUACCAAGU CUCAGGGGAA ACUUUGAGAU GGCCUUGCAA A GGGUAUGG ...String:
GGCCGGGUGG AGGGAAAAGU UAUCAGGCAU GCACCUGGUA GCUAGUCUUU AAACCAAUAG AUUGCAUCGG UUUAAAAGGC AAGACCGUC AAAUUGCGGG AAAGGGGUCA ACAGCCGUUC AGUACCAAGU CUCAGGGGAA ACUUUGAGAU GGCCUUGCAA A GGGUAUGG UAAUAAGCUG ACGGACAUGG UCCUAACCAC GCAGCCAAGU CCUAAGUCAA GGAUGGUUCU UGAUAUGGAU GC AGUUCAC AGACUAAAUG UCGGUCGGGG AAGAGAACCA UCCUCUUCUC AUAAGAUAUA GUCGGACCUC UCCUUAAUGG GAG CUAGCG GAUGAAGUGA UGCAACACUG GAGCCGCUGG GAACUAAUUU GUAUGCGAA

GENBANK: GENBANK: V01416.1

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Macromolecule #2: Group I intron, 3 prime fragment plus 3 prime exon

MacromoleculeName: Group I intron, 3 prime fragment plus 3 prime exon / type: other / ID: 2 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 11.803012 KDa
SequenceString:
AGUAUAUUGA UUAGUUUUGG AGUACUC(DG)(DA)C CCGGCCA

GENBANK: GENBANK: V01416.1

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 20 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-acetateTris-acetate
30.0 mMMgCl2Magnesium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 82575

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