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- PDB-9c6i: Cryo-EM structure of a group IIC intron RNA -

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Basic information

Entry
Database: PDB / ID: 9c6i
TitleCryo-EM structure of a group IIC intron RNA
ComponentsGroup IIC Intron
KeywordsRNA / splicing / ribozyme
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesOceanobacillus iheyensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.56 Å
AuthorsToor, N.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141706 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122532 United States
CitationJournal: Nat Commun / Year: 2025
Title: Scaffold-enabled high-resolution cryo-EM structure determination of RNA.
Authors: Daniel B Haack / Boris Rudolfs / Shouhong Jin / Alexandra Khitun / Kevin M Weeks / Navtej Toor /
Abstract: Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are ...Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA. We demonstrate this technology by determining the structures of the 86-nucleotide (nt) thiamine pyrophosphate (TPP) riboswitch aptamer domain and the recently described 210-nt raiA bacterial non-coding RNA involved in sporulation and biofilm formation. In the case of the TPP riboswitch aptamer domain, the scaffolding approach allowed visualization of the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch adopts an open Y-shaped conformation in the absence of ligand. Using this scaffold approach, we determined the structure of raiA at 2.5 Å in the core. Our versatile scaffolding strategy enables efficient RNA structure determination for a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.
History
DepositionJun 7, 2024Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Group IIC Intron
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,73922
Polymers135,2291
Non-polymers51021
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chain Group IIC Intron


Mass: 135229.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oceanobacillus iheyensis (bacteria) / Production host: Escherichia coli (E. coli)
#2: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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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: Group IIC Intron / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: .135 MDa / Experimental value: NO
Source (natural)Organism: Oceanobacillus iheyensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 382753 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0089470
ELECTRON MICROSCOPYf_angle_d0.67714773
ELECTRON MICROSCOPYf_dihedral_angle_d16.4314713
ELECTRON MICROSCOPYf_chiral_restr0.0431965
ELECTRON MICROSCOPYf_plane_restr0.005393

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