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- EMDB-13926: Twist-corrected RNA origami 5-helix Tile A -

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Basic information

Entry
Database: EMDB / ID: EMD-13926
TitleTwist-corrected RNA origami 5-helix Tile A
Map dataSharpened map with b factor of -252.
Sample
  • Complex: Twist-corrected 5-helix tile A.
    • RNA: Chains: Q
KeywordsRNA / Origami / nanostructure
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.14 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
DepositionNov 30, 2021-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13926.png

Downloads & links

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Map

FileDownload / File: emd_13926.map.gz / Format: CCP4 / Size: 233.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map with b factor of -252.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.29 Å/pix.
x 394 pix.
= 509.836 Å		1.29 Å/pix.
x 394 pix.
= 509.836 Å		1.29 Å/pix.
x 394 pix.
= 509.836 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.294 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.138
Minimum - Maximum-0.21351494 - 0.6547129
Average (Standard dev.)-0.00023263601 (±0.012246599)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions394394394
Spacing394394394
CellA=B=C: 509.836 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13926_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_13926_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_13926_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Twist-corrected 5-helix tile A.

EntireName: Twist-corrected 5-helix tile A.
Components
  • Complex: Twist-corrected 5-helix tile A.
    • RNA: Chains: Q

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Supramolecule #1: Twist-corrected 5-helix tile A.

SupramoleculeName: Twist-corrected 5-helix tile A. / 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: 175 KDa

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Macromolecule #1: Chains: Q

MacromoleculeName: Chains: Q / type: rna / ID: 1 / Details: In vitro transcribed RNA / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 177.720031 KDa
SequenceString: GGGCACUUAC CCUUUAGUGC GAAGGUUUCG ACCUUCGAUC CAUUUGUUCG CAAAUGGGAU GAUCUUCGGA UCAUCCGCGU AGUCUGUUC AGUCGUUUCG ACGACUGGCC CCACUUCGGU GGGGCCACGG UACUUAGAAG UGAACACUAA GUGUCGUGAA C ACCAUUUG ...String:
GGGCACUUAC CCUUUAGUGC GAAGGUUUCG ACCUUCGAUC CAUUUGUUCG CAAAUGGGAU GAUCUUCGGA UCAUCCGCGU AGUCUGUUC AGUCGUUUCG ACGACUGGCC CCACUUCGGU GGGGCCACGG UACUUAGAAG UGAACACUAA GUGUCGUGAA C ACCAUUUG GUUAACUGCU CAAACUAAAU GGUGAUGAGG GAAGGAAUGA CCCUCAUCGG ACUACGCGAU CCGAGUGAUG GG AAUGGCU GACCCAUCGC UCGGCACUGG AGGGUGAGUG CCCCUCAUUC GCAUAAGGGC CGACCCAGAC AACAGCCAAG UUU GGGUCG GAGAUGCGAA CAUUCCACGC AUCUGAACGG UUGAGAACUU ACAAGGGCAA GAGCAGAGUC CUUGUAAGGG CUUU ACACG UCAAGUUCAC AGACGUGUAA GGCCCGUCGC CCUUCGGGGC GACGUUCACG GCAUUUCGAU GCCGUGCAGC CUGUU CGCA GGCUGCUUGA CCGUUCCCCU GCCCUUUCGA GGGCAGACUA CUCUUCGGAG UAGUCUUAUG UGAAUGAG

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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

Concentration3.0 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.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: 15mA current on a glocube.
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
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6122 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2 / Details: Images were collected as 56 frame movies.
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 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

Particle selectionNumber selected: 605848
Details: Particle picking was performed using the template picker from cryoSPARC with templates generated from an ab initio model from the same dataset.
Startup modelType of model: NONE
Details: Initial volume was generated using a 3 class ab initio reconstruction given 30000 randomly selected particles.
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 5.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 166751
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Details: Initial angle assignment was from heterogeneous refinement into 3 classes in cryoSPARC
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 3D classificationNumber classes: 50 / Avg.num./class: 5000 / Software - Name: cryoSPARC (ver. 3.2.0)
Details: A 2D classification was performed on 183420 particles and obvious junk classes were discarded. This led to a nominal improvement in the GFSC (0.01A) but significant improvement in the map quality.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation Coefficient
Output model

PDB-7qdu:
Twist-corrected RNA origami 5-helix Tile A

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