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- EMDB-13625: Mature conformer 2 of a 6-helix bundle of RNA with a clasp -

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Basic information

Entry
Database: EMDB / ID: EMD-13625
TitleMature conformer 2 of a 6-helix bundle of RNA with a clasp
Map data
Sample
  • Complex: 6-helix bundle of RNA with clasp
    • RNA: 6HBC-mature
KeywordsRNA / origami / nanostructure
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.43 Å
AuthorsMcRae EKS / Bogglid A / Boesen T / Andersen ES
Funding support Denmark, 1 items
OrganizationGrant numberCountry
Danish Council for Independent Research31789 Denmark
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
DepositionSep 27, 2021-
Header (metadata) releaseOct 5, 2022-
Map releaseOct 5, 2022-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13625.png

Downloads & links

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Map

FileDownload / File: emd_13625.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.68 Å
Density
Contour LevelBy AUTHOR: 0.33
Minimum - Maximum-0.102759026 - 0.788604
Average (Standard dev.)0.0004021263 (±0.028645564)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 430.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13625_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map sharpened with a bfactor of 420 in cryosparc

Fileemd_13625_additional_1.map
AnnotationMap sharpened with a bfactor of 420 in cryosparc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13625_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13625_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 6-helix bundle of RNA with clasp

EntireName: 6-helix bundle of RNA with clasp
Components
  • Complex: 6-helix bundle of RNA with clasp
    • RNA: 6HBC-mature

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Supramolecule #1: 6-helix bundle of RNA with clasp

SupramoleculeName: 6-helix bundle of RNA with clasp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: In vitro transcribed RNA purified by SEC. This is the mature conformer.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 232 KDa

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Macromolecule #1: 6HBC-mature

MacromoleculeName: 6HBC-mature / type: rna / ID: 1
Source (natural)Organism: synthetic construct (others)
SequenceString: GGGAGAGUAC UAUUCAGAUG CAGACCGCAA GUUCAGAGCG GUUUGCAUCU AGGGUACGUU UUCGAACGUA UCCUCCGACU AAGUGUAUUC GUAUACUUAG UGCCUUGUGC CUGCUUCGGC AGGCAUGACC CAAAUGUGCC UUUCGGGGCA CAUUUCCGGU CAUCCAAGUU ...String:
GGGAGAGUAC UAUUCAGAUG CAGACCGCAA GUUCAGAGCG GUUUGCAUCU AGGGUACGUU UUCGAACGUA UCCUCCGACU AAGUGUAUUC GUAUACUUAG UGCCUUGUGC CUGCUUCGGC AGGCAUGACC CAAAUGUGCC UUUCGGGGCA CAUUUCCGGU CAUCCAAGUU CGCUUGGGUG AUGCGGGCGU AUAGGUUCGU CUAUACGUCC GCGUUUUCCG AGAAGAGGUA ACUCGGGAAA CCGGUCCACG UGACAAAGGU AGAGUUACGU GGAGGGAGCA GCUGCAAAGG GAUAAUGCAG UUGCUGGCUG GAUGCCAGAA CUCACGACUG GCAUCUACGG GGAUGGUGCU CUCCCAAUUC UCCAUUUACC GCCGAAUCGA CCCCAACGUG AGAGGGGUCG GUUCCCCGAG CAUAGACCAA UAUCCCAGGU UUAUGCUCCC CAACGCUGGA CGAACUACCU ACGUCUAGCG UUCCGGCAAA UGAGUCAAUA CCUCAGACUU AUUUGCGGUG CCUGAGCCUA AACUGAACAU GGGUUCAGGC AUCUUGGCUC CAGUUCGCUG GAGCCGACGG UAGCGCUGCG UUCGCGCAGU GCUAGGGAGC AUCCGUUUUC GAGCGGAUGC UGGGCGGUUG CCUGUUCGCA GGCAAUCGGG CCUACUCAUG AUUCGUCAUG AGUGGUGACA GCGUGAUGUU CGCAUUACGC UGUCGGGUAG AUGGAGAAUU

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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

Concentration2.8 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMHEPES
5.0 mMMgCl2
100.0 mMKCl

Details: Freshly prepared and filtered through 0.22 micron filter prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 294 K / Instrument: LEICA EM GP
Details: 3 microlitre 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. Sample was then stored at -20 for three weeks before grid preparation.

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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: 0.03508 mm / Nominal magnification: 130000
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 K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2658 / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 332956
Details: For the final particle set, Topaz particle picker was used within cryoSPARC with the pre-trained ResNet16(64 units) model and a downsampling 4-fold..
Startup modelType of model: NONE
Details: Initial volume was generated using a 4 class ab initio reconstruction given 40000 randomly selected particles.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The entire set of 332956 particles were then 3D classified into one of the four initial ab initio volumes.
Final 3D classificationNumber classes: 4 / Avg.num./class: 50000 / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The final classification was a heterogeneous refinement from 4 different ab initio volumes and 201745 particles.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The final angle assignment was determined using the local refinement beta job and a mask covering the entire volume.
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 7.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 55657
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementOverall B value: 420

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