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- PDB-8btz: Single-stranded Paranemic Crossover RNA Triangle (PXT) -

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Basic information

Entry
Database: PDB / ID: 8btz
TitleSingle-stranded Paranemic Crossover RNA Triangle (PXT)
ComponentsRNA Paranemic croosover triangle (PXT)
KeywordsRNA / Cotranscriptional folding / RNA origami / RNA nanotechnology / Paranemic crossover
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.39 Å
AuthorsSampedro, N. / McRae, E.K.S. / Andersen, E.S.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Danish Council for Independent Research31789 Denmark
CitationJournal: Small / Year: 2023
Title: An RNA Paranemic Crossover Triangle as A 3D Module for Cotranscriptional Nanoassembly.
Authors: Néstor Sampedro Vallina / Ewan K S McRae / Cody Geary / Ebbe Sloth Andersen /
Abstract: RNA nanotechnology takes advantage of structural modularity to build self-assembling nano-architectures with applications in medicine and synthetic biology. The use of paranemic motifs, that form ...RNA nanotechnology takes advantage of structural modularity to build self-assembling nano-architectures with applications in medicine and synthetic biology. The use of paranemic motifs, that form without unfolding existing secondary structure, allows for the creation of RNA nanostructures that are compatible with cotranscriptional folding in vitro and in vivo. In previous work, kissing-loop (KL) motifs have been widely used to design RNA nanostructures that fold cotranscriptionally. However, the paranemic crossover (PX) motif has not yet been explored for cotranscriptional RNA origami architectures and information about the structural geometry of the motif is unknown. Here, a six base pair-wide paranemic RNA interaction that arranges double helices in a perpendicular manner is introduced, allowing for the generation of a new and versatile building block: the paranemic-crossover triangle (PXT). The PXT is self-assembled by cotranscriptional folding and characterized by cryogenic electron microscopy, revealing for the first time an RNA PX interaction in high structural detail. The PXT is used as a building block for the construction of multimers that form filaments and rings and a duplicated PXT motif is used as a building block to self-assemble cubic structures, demonstrating the PXT as a rigid self-folding domain for the development of wireframe RNA origami architectures.
History
DepositionNov 30, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA Paranemic croosover triangle (PXT)


Theoretical massNumber of molelcules
Total (without water)76,3941
Polymers76,3941
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA Paranemic croosover triangle (PXT)


Mass: 76393.867 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: In vitro transcribed RNA / Source: (synth.) synthetic construct (others)

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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: Enzymatically synthesized and isothermally folded single stranded RNA origami
Type: COMPLEX / Details: In vitro transcribed RNA purified by SEC. / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 76.38 kDa/nm / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.8 / Details: Filtered through 0.22 micron filter
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
25 mMMagnesium ChlorideMgCl21
350 mMPotassium ChlorideKCl1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was purified by size exclusion chromatography and concentrated in an Amicon spin concentrator.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 294 K
Details: 3 microliter droplet, 4 second delay before blotting, 6 second blot, 0 second delay before plunging.

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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 / Calibrated magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
UCSF ChimeraX1.3/v9model building
EM software
IDNameVersionCategoryDetails
2EPUimage acquisitionAFIS data collection
7UCSF ChimeraX1.2model fitting
8ISOLDE1.2model fitting
13cryoSPARC3.2.03D reconstruction
14PHENIX1.19.2-4158-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 5.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 437831 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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