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- EMDB-13630: Mature conformer of a 6-helix bundle of RNA with clasp -

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Basic information

Entry
Database: EMDB / ID: EMD-13630
TitleMature conformer of a 6-helix bundle of RNA with clasp
Map data
Sample
  • Complex: Mature conformer of a 6-helix bundle of RNA with clasp
    • RNA: Chains: B
KeywordsRNA / origami / nanostructure
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 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_13630.png

Downloads & links

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Map

FileDownload / File: emd_13630.map.gz / Format: CCP4 / Size: 111.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.18846256 - 1.3412237
Average (Standard dev.)-0.0015745665 (±0.046692878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions308308308
Spacing308308308
CellA=B=C: 406.56003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13630_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: Sharpened map from cyroSPARC with bfactor of 250.

Fileemd_13630_additional_1.map
AnnotationSharpened map from cyroSPARC with bfactor of 250.
Projections & Slices
AxesZYX

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Histogram

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Half map: #2

Fileemd_13630_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_13630_half_map_2.map
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Sample components

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Entire : Mature conformer of a 6-helix bundle of RNA with clasp

EntireName: Mature conformer of a 6-helix bundle of RNA with clasp
Components
  • Complex: Mature conformer of a 6-helix bundle of RNA with clasp
    • RNA: Chains: B

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

SupramoleculeName: Mature conformer of a 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: Chains: B

MacromoleculeName: Chains: B / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 231.958125 KDa
SequenceString: GGGAGAGUAC UAUUCAGAUG CAGACCGCAA GUUCAGAGCG GUUUGCAUCU AGGGUACGUU UUCGAACGUA UCCUCCGACU AAGUGUAUU CGUAUACUUA GUGCCUUGUG CCUGCUUCGG CAGGCAUGAC CCAAAUGUGC CUUUCGGGGC ACAUUUCCGG U CAUCCAAG ...String:
GGGAGAGUAC UAUUCAGAUG CAGACCGCAA GUUCAGAGCG GUUUGCAUCU AGGGUACGUU UUCGAACGUA UCCUCCGACU AAGUGUAUU CGUAUACUUA GUGCCUUGUG CCUGCUUCGG CAGGCAUGAC CCAAAUGUGC CUUUCGGGGC ACAUUUCCGG U CAUCCAAG UUCGCUUGGG UGAUGCGGGC GUAUAGGUUC GUCUAUACGU CCGCGUUUUC CGAGAAGAGG UAACUCGGGA AA CCGGUCC ACGUGACAAA GGUAGAGUUA CGUGGAGGGA GCAGCUGCAA AGGGAUAAUG CAGUUGCUGG CUGGAUGCCA GAA CUCACG ACUGGCAUCU ACGGGGAUGG UGCUCUCCCA AUUCUCCAUU UACCGCCGAA UCGACCCCAA CGUGAGAGGG GUCG GUUCC CCGAGCAUAG ACCAAUAUCC CAGGUUUAUG CUCCCCAACG CUGGACGAAC UACCUACGUC UAGCGUUCCG GCAAA UGAG UCAAUACCUC AGACUUAUUU GCGGUGCCUG AGCCUAAACU GAACAUGGGU UCAGGCAUCU UGGCUCCAGU UCGCUG GAG CCGACGGUAG CGCUGCGUUC GCGCAGUGCU AGGGAGCAUC CGUUUUCGAG CGGAUGCUGG GCGGUUGCCU GUUCGCA GG CAAUCGGGCC UACUCAUGAU UCGUCAUGAG UGGUGACAGC GUGAUGUUCG CAUUACGCUG UCGGGUAGAU GGAGAAUU

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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

Concentration2.3 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMHEPES
5.0 mMMagnesium ChlorideMgCl2
100.0 mMPotassium ChlorideKCl

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 defocus max: 2.5 µm / Nominal defocus min: 0.75 µm / 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: 8359 / 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: 1476537
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 6 class ab initio reconstruction given 100000 randomly selected particles.
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 3D classificationNumber classes: 3 / Avg.num./class: 110000 / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The final classification was a heterogeneous refinement from 4 different ab initio volumes and 417569 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: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 337099
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2d1b

DetailsHelical templates and tetraloops were generated in RNAbuild. Kissing loops were modelling based on the PDB:2D1B structure. A combination of PHENIX RSR and ISOLDE were used to arrive at the final structure.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 303 / Target criteria: Correlation Coefficient
Output model

PDB-7ptl:
Mature conformer of a 6-helix bundle of RNA with clasp

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