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- EMDB-16245: Double-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT) -

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Basic information

Entry
Database: EMDB / ID: EMD-16245
TitleDouble-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT)
Map data
Sample
  • Complex: Double-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT)
    • RNA: RNA (354-MER)
KeywordsRNA origami / Nanostructure / self-assembly / RNA
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.56 Å
AuthorsSampedro N / Andersen ES / McRae EKS
Funding support Denmark, 1 items
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, 2022-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16245.png

Downloads & links

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Map

FileDownload / File: emd_16245.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.62 Å
Density
Contour LevelBy AUTHOR: 0.0104
Minimum - Maximum-0.21093555 - 0.7021365
Average (Standard dev.)-0.00016296507 (±0.015015641)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 414.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-A

Fileemd_16245_half_map_1.map
AnnotationHalf-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-B

Fileemd_16245_half_map_2.map
AnnotationHalf-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Double-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT)

EntireName: Double-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT)
Components
  • Complex: Double-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT)
    • RNA: RNA (354-MER)

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Supramolecule #1: Double-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT)

SupramoleculeName: Double-motif Single-stranded Paranemic Crossover RNA Triangle (2PXT)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: In vitro transcribed RNA purified by SEC.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 113.63 kDa/nm

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Macromolecule #1: RNA (354-MER)

MacromoleculeName: RNA (354-MER) / type: rna / ID: 1 / Details: In vitro transcribed RNA / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 113.647586 KDa
SequenceString: GUUACUCACC UCUUCGGAGA AAACGGUCCG AGCGUGGUUC GCCACUAAAA UACCGUUUUG GCCUGAAUGU GUCUGGUCCU UCCGCAGCC GUUUGAUGAG UUCGCUCAUC AUAGCGGGCU UACCAAACAG UUCGCUGCCA CCGUGCGGAA GCAGUUUCGA C UGAUAGAU ...String:
GUUACUCACC UCUUCGGAGA AAACGGUCCG AGCGUGGUUC GCCACUAAAA UACCGUUUUG GCCUGAAUGU GUCUGGUCCU UCCGCAGCC GUUUGAUGAG UUCGCUCAUC AUAGCGGGCU UACCAAACAG UUCGCUGCCA CCGUGCGGAA GCAGUUUCGA C UGAUAGAU CACGGUGGGA CUAUUCGUAG UCUUUGGUGA GCUCGUUACG UUUCUUCGGA AACGAACGGC UGAUCUAUGA CC AGAUGUA UUCAGGCCGU GAGUAACGUC GCCGUACAUC UUCGGAUGUA GCGUCCCUAU UUUACAAACU UCGGUUUGGC UCG GAGGGA CGCAGCAGGG UUCGCCCUGC UCGGCGAC

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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

BufferpH: 7.8
Component:
ConcentrationNameFormula
25.0 mMHEPES
5.0 mMMagnesium ChlorideMgCl2
50.0 mMPotassium ChlorideKCl
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 294 K
Details: 3 microliter droplet, 4 second delay before blotting, 6 second blot, 0 second delay before plunging..
DetailsSample was purified by size exclusion chromatography and concentrated in an Amicon spin concentrator.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.56 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 128507
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)

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