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- EMDB-28683: Single-Molecule 3D Image of 16 Helix RNA Origami Satellite by Ind... -

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Entry
Database: EMDB / ID: EMD-28683
TitleSingle-Molecule 3D Image of 16 Helix RNA Origami Satellite by Individual Particle Electron Tomography (No. 15)
Map data3D Image of 16 Helix RNA Origami Satellite by Individual Particle Electron Tomography (No. 15)
Sample
  • Complex: 16 Helix RNA origami satellite
    • RNA: 16 Helix RNA origami satellite
KeywordsRNA / origami / Individual Particle Electron Tomography / Single-Molecule 3D Image
Biological speciesunidentified (others)
Methodelectron tomography / cryo EM / Resolution: 26.0 Å
AuthorsLiu J / Ren G
Funding support United States, 5 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-AC02-05CH11231 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL115153 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM104427 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01MH077303 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK042667 United States
CitationJournal: Nat Nanotechnol / Year: 2023
Title: Structure, folding and flexibility of co-transcriptional RNA origami.
Authors: Ewan K S McRae / Helena Østergaard Rasmussen / Jianfang Liu / Andreas Bøggild / Michael T A Nguyen / Nestor Sampedro Vallina / Thomas Boesen / Jan Skov Pedersen / Gang Ren / Cody Geary / ...Authors: Ewan K S McRae / Helena Østergaard Rasmussen / Jianfang Liu / Andreas Bøggild / Michael T A Nguyen / Nestor Sampedro Vallina / Thomas Boesen / Jan Skov Pedersen / Gang Ren / Cody Geary / Ebbe Sloth Andersen /
Abstract: RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the ...RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.
History
DepositionOct 26, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28683.png

Downloads & links

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Map

FileDownload / File: emd_28683.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D Image of 16 Helix RNA Origami Satellite by Individual Particle Electron Tomography (No. 15)
Voxel sizeX=Y=Z: 2.1 Å
Density
Minimum - Maximum-0.4556731 - 2.8340843
Average (Standard dev.)0.02744751 (±0.16666634)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 537.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : 16 Helix RNA origami satellite

EntireName: 16 Helix RNA origami satellite
Components
  • Complex: 16 Helix RNA origami satellite
    • RNA: 16 Helix RNA origami satellite

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Supramolecule #1: 16 Helix RNA origami satellite

SupramoleculeName: 16 Helix RNA origami satellite / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: In vitro purified RNA.
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 590 KDa

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Macromolecule #1: 16 Helix RNA origami satellite

MacromoleculeName: 16 Helix RNA origami satellite / type: rna / ID: 1
SequenceString: GGAACAAGCA UAACCCGUCC AGGUUCUCAA GACCAAAGAG AACUUGGACC UAUAGUCGGC GUCUACCCGU GUCGCCACGG GUGGACGCCG GCUAUAGGCU UCCCUUUCAU CGCACCUCGC GUUCGCACGC GGGGUGCGCU GACGGUGGGG CUAGCGCCCC AUCGUCAGCC ...String:
GGAACAAGCA UAACCCGUCC AGGUUCUCAA GACCAAAGAG AACUUGGACC UAUAGUCGGC GUCUACCCGU GUCGCCACGG GUGGACGCCG GCUAUAGGCU UCCCUUUCAU CGCACCUCGC GUUCGCACGC GGGGUGCGCU GACGGUGGGG CUAGCGCCCC AUCGUCAGCC CUAUCCCGUU ACUUUACAGA GGUCCCGCGC AAUUUGUGGA CCUUGGCAGG UCCGCAAAUU GCGUGGGACG GUGCGGGGUU UCAUGGCUGA AACCUCGCAC CGUAAUAGCG GUGAUUAACG AGCUAGCGCU CGUUGAUCAC CCACUUCGGU AACUCAAGAA CCGGAGUGCG UUUGGAUCUU CAAUCCUAGA GAAGGUCCAA ACGGCUGUUA CCUCUGUGAA GUAACGACGC GUCACAAUGG ACUAGUGAUG CGUCGCACGC UACCCCAAAA CCAGAGGGGU GGCGUGCGGG GUAGGGAUGA AGGGGAAGCG GGUUGUGUUU GUUCCCAAAU ACCUUCUUCG GGCCUCUCUU CCAUGAUACU AUUGAUUAUU UCGCUUCUAA UAUACCCCGU AGUCUCCGGA CUACGCCCGA CGUCCGCGUC GGGGAGGGGC UAACAGACUA AGCUCCUCGG CCCGUAAUAC CGAAACGGGG UAAUACCCAC CCGCAGUUCC CGGGACUGUA AUCGGUAAAC AGUCGGCGCA AAGUCUGAGC GCCCUUACUC UUCGGAGUAA GCCGGCAGUA UACGUACUGC CCGAGGGUGC UCGUAUUUGU GCGCAAAUAC CGCAUAUGGU ACGCCAUAUG GGAGUGGUGA CUUUCGAGUC AUCACUCCCA CGUUUGAAGG AUGAACAAGC GUGCGCCCUU GAAAUCGUCA CAAGGGGAGC GCCCUCGGGG AAUUGCGGCG GUCACAAGAC GAUAGUGACG GCCCAAUCAU CCAGGGCCCC AACUCUACGG AGUUGGCGCU AGUCUUACGA GACUAGGUGG GUGUUACGUA UAUUGGAAGC GAGAUAAUCA AUGGUAUCCU GGUUAAUGGA GGAGAGGGGC GUUUUAUAGU GAAAGUCCAA CACUAUGAAA CCGGGCUCCG GGAUCAACUA GGAAGAUCUC GGAGCGGCGC AUUUUCUAUU CGCCUUCUCU UACAUAUCUA AAUACUCUUU UCUCGCGCUG GUAUCCGUAC CAGCCGGAUG AUACGUCAUC CGCAGCUGAG AAUAGGUGAC UCAGCUGGCG CGGAAAUCCU GUAUCCGCCU UACUUCCGCC UACGCUCUAG GCGUGGAUAA ACAGGAAAUC CACGACCCAA CACCUAAGGG UCGAGCAAUU CCGAUUGCUC GCCCUCGAAU ACGUUCGAGG CUGGCGUGCU GGGUGGUGUC CGCACCACCG GGAUCCUGUA CGCAGGAUCG GCGAGGAGGG UUUCGACCCU UCUCGCCGAG UGAAUAACUG UUGAAUUCGC UCCCCGUCCC AAGUCUUGAG GGACGCAGCA UGCCAGCUAG GGCGUAGCGG CCUUAACAAG ACAAAGGCGA GGUAACAACA GAACCUCCCC UGUCUGCGGA CAGGGCGGUU GCUCUCCGGA GCAACGCGGA GGUAAGGAGA GAAGAGUGUU UAGAUGUGUA AGAGGAGGCG CUUGAGAAAU AGAGAAUGCC AUAGCCGUAU GUAAAUUGGU CAUACAUAUG GCUAUGGGUC UGCAGUUACG ACUGCGGACC GCCCUGAUGU AGGUACGCCU AUAUCAGCCG GGCUGCUCCG UACGCGGAGU AGCCGCCGUC GGGUGGCUAC GGCCAUCCGA CCCCGCAGUC AUGAUUCGUC AUGGCUGCCA GACGUGCGGA UUACGAUCCG UACGUCUGGA AGGAGGUGUU UG

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration1.8 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMHEPES
5.0 mMMgCl2MgCl2
100.0 mMKClKCl
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: LEICA EM GP
DetailsIn vitro transcribed RNA was purified by size exclusion chromatography and spin concentrated in amicon spin columns.
SectioningOther: NO SECTIONING

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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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 6.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IPET
Details: The targeted images of individual particle were reconstructed by Individual-Particle Electron Tomography (IPET). To reduce the missing-wedge artifact caused by the limited tilt angle range, ...Details: The targeted images of individual particle were reconstructed by Individual-Particle Electron Tomography (IPET). To reduce the missing-wedge artifact caused by the limited tilt angle range, the final 3D map was submitted to a low-tilt tomographic 3D reconstruction method (LoTToR). IPET 3D map was low-pass filtered to 6 nm, and displayed by Chimera with applied the hidden dust function.
Number images used: 11
DetailsMotion correction of the multi-frame movie was conducted by MotionCor2. The tilt series of whole micrographs were initial aligned by IMOD. Additionally, to reduce the image noise, tilt series were further conducted by a machine learning, a median-filter process and a contrast enhancement method.
FSC plot (resolution estimation)

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