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- EMDB-16244: Single-stranded Paranemic Crossover RNA Triangle (PXT) -

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Basic information

Entry
Database: EMDB / ID: EMD-16244
TitleSingle-stranded Paranemic Crossover RNA Triangle (PXT)
Map data
Sample
  • Complex: Enzymatically synthesized and isothermally folded single stranded RNA origami
    • RNA: RNA Paranemic croosover triangle (PXT)
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.39 Å
AuthorsSampedro N / McRae EKS / Andersen ES
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) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateApr 5, 2023-
Current statusApr 5, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16244.png

Downloads & links

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Map

FileDownload / File: emd_16244.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.41 Å/pix.
x 128 pix.
= 180.48 Å		1.41 Å/pix.
x 128 pix.
= 180.48 Å		1.41 Å/pix.
x 128 pix.
= 180.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.41 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.124
Minimum - Maximum-0.16785075 - 0.6370303
Average (Standard dev.)0.0028441949 (±0.027258769)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 180.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_16244_half_map_1.map
AnnotationHalf_map_A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_16244_half_map_2.map
AnnotationHalf_map_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 : Enzymatically synthesized and isothermally folded single stranded...

EntireName: Enzymatically synthesized and isothermally folded single stranded RNA origami
Components
  • Complex: Enzymatically synthesized and isothermally folded single stranded RNA origami
    • RNA: RNA Paranemic croosover triangle (PXT)

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Supramolecule #1: Enzymatically synthesized and isothermally folded single stranded...

SupramoleculeName: Enzymatically synthesized and isothermally folded single stranded RNA origami
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all / Details: In vitro transcribed RNA purified by SEC.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 76.38 kDa/nm

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Macromolecule #1: RNA Paranemic croosover triangle (PXT)

MacromoleculeName: RNA Paranemic croosover triangle (PXT) / type: rna / ID: 1 / Details: In vitro transcribed RNA / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 76.393867 KDa
SequenceString: GGAAUAUCGU CAUGGUGAUU CGUCACCAUG AGGCUAGAUC UCAUAUCUAG CGCUUUCGAG CGCUAGAGUC CUUAUCUAGC CGGUUUAUA CUUUCGAGUG UGAACCCGAU AUUCCGCGGA UCACUAUGAG UCGUUCGCGG CUCAUAGUCC GGCUCAAAGG A CAUCAUGG ...String:
GGAAUAUCGU CAUGGUGAUU CGUCACCAUG AGGCUAGAUC UCAUAUCUAG CGCUUUCGAG CGCUAGAGUC CUUAUCUAGC CGGUUUAUA CUUUCGAGUG UGAACCCGAU AUUCCGCGGA UCACUAUGAG UCGUUCGCGG CUCAUAGUCC GGCUCAAAGG A CAUCAUGG CCUGUUCGCA GGUUGUGAUU AUGAGUGAGC CGGGUAAGGC AUACCGUUCG CGGUAUGUCU UACGAUCCGC

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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

Concentration3 mg/mL
BufferpH: 7.8
Component:
ConcentrationNameFormula
25.0 mMHEPES
5.0 mMMagnesium ChlorideMgCl2
50.0 mMPotassium ChlorideKCl

Details: Filtered through 0.22 micron filter
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 294 K / Instrument: LEICA EM GP
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 5.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 437831

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

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

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