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- EMDB-13627: 6-Helix bundle of RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-13627
Title6-Helix bundle of RNA
Map data
Sample
  • Complex: 6-helix bundle of RNA
    • RNA: 6HB
KeywordsRNA / origami / nanostructure
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.61 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_13627.png

Downloads & links

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Map

FileDownload / File: emd_13627.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.59 Å
Density
Contour LevelBy AUTHOR: 0.36
Minimum - Maximum-0.13124888 - 0.8335619
Average (Standard dev.)0.0023202929 (±0.039802928)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 331.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13627_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map from cryoSPARC with bfactor of 316

Fileemd_13627_additional_1.map
AnnotationSharpened map from cryoSPARC with bfactor of 316
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13627_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13627_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : 6-helix bundle of RNA

EntireName: 6-helix bundle of RNA
Components
  • Complex: 6-helix bundle of RNA
    • RNA: 6HB

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Supramolecule #1: 6-helix bundle of RNA

SupramoleculeName: 6-helix bundle of RNA / 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: 232 KDa

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Macromolecule #1: 6HB

MacromoleculeName: 6HB / type: rna / ID: 1 / Details: In vitro transcribed RNA
Source (natural)Organism: synthetic construct (others)
SequenceString: GGGAAAUCCC GCCCUGAUAC GGUUCACGUU CGCGUGGACC GUCGGGUGGU CCGCUUACGA GCGGGCCACG GCGACCGUGC AAUGCGUUGC AUGGUCCGGG CUUGCUCGCU ACGGCGAGUA AGCGGGGAAC UAGAGGUGCG CCUCUGGUUC GGCGCUAUGU GGCUCCGGCU ...String:
GGGAAAUCCC GCCCUGAUAC GGUUCACGUU CGCGUGGACC GUCGGGUGGU CCGCUUACGA GCGGGCCACG GCGACCGUGC AAUGCGUUGC AUGGUCCGGG CUUGCUCGCU ACGGCGAGUA AGCGGGGAAC UAGAGGUGCG CCUCUGGUUC GGCGCUAUGU GGCUCCGGCU ACAUAGUGCC CCGAUUGGGG AACCCCUAAC CCCAGUCGGC CCGUUGGGUC ACAACGGUUC AGUGAUCCAA CCCGGGUAUG GCACAACGCG AUAGUGCUAU ACCGCCGUAU GUUCGGAAUG AUGGACCGAG CAUACCCGCA CGUGGUGGAA CACUCGACCA CUACGUGAUU AGGGUGGGGU UUCCCAGAAU UUGUAUCGUG GCCGACUGAG AACUAACGAG UGAAGUUCUU AGUCCCCGUU UUGAGAUGAA CCAUCAACAU CUCGAAACCC GGAUAUCGGU GUAAAUCGCG AACACCGGUA UCGCCGAGCU AACUGUAAGA ACCGAACAGU UGGCUCCAUG UUUAAACGGC AAUAGGGGAG CUGUUUAGAC AUGAGCAGCG GCGUUCGCGC CGUUGCUGGC GUCCGUUUGA UCCGUCAAAU GGACCGGGCA GUUUAUCUCC GGAUAAGCUG CGGGGUCCGA UGAUUCCGAU CAUUGGACGG CGUCGGGUUG GUGCGCCAAC UCGACUGAUC CUUUUACCUA CGGGUAGAAG GAUCACAUGA UGCAAGUUCU

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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

Concentration2.5 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.

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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 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 K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 2244 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2 / Details: Images were collected as 56 frame movies.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 258034
Details: Particles were picked with templated particle pickign within CS-Live
Startup modelType of model: NONE
Details: Initial volume was generated using a 3 class ab initio reconstruction given 50516 randomly selected particles.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The entire set of 258034 particles were then 3D classified into one of the three initial ab initio volumes.
Final 3D classificationNumber classes: 3 / Avg.num./class: 80000 / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The final classification was a heterogeneous refinement from 3 different ab initio volumes and 258034 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: 6.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 92383
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementOverall B value: 316

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