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- PDB-7ptk: Young conformer of a 6-helix bundle of RNA with clasp -

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Basic information

Entry
Database: PDB / ID: 7ptk
TitleYoung conformer of a 6-helix bundle of RNA with clasp
ComponentsRNA
KeywordsRNA / origami / nanostructure
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.18 Å
AuthorsMcRae, E.K.S. / Andersen, E.S.
Funding support Denmark, 1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 29, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 2, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RNA


Theoretical massNumber of molelcules
Total (without water)231,9581
Polymers231,9581
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area120260 Å2

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Components

#1: RNA chain RNA /


Mass: 231958.125 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: In vitro transcribed RNA / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Young conformer of a 6-helix bundle of RNA with clasp / Type: COMPLEX
Details: In vitro transcribed RNA purified by SEC. This is the young conformer.
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.232 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Buffer solutionpH: 8
Details: Freshly prepared and filtered through 0.22 micron filter prior to use.
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
25 mMMagnesium ChlorideMgCl21
3100 mMPotassium ChlorideKCl1
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample 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.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 294 K
Details: 3 microlitre droplet, 4 second delay before blotting, 6 second blot, 0 second delay before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 17975 / Details: Images were collected as 56 frame movies.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2.0particle selection
2EPUimage acquisitionAFIS data collection
4cryoSPARC3.2.0CTF correction
7UCSF ChimeraX1.2model fitting
8ISOLDE1.2model fitting
10cryoSPARC3.2.0initial Euler assignment
11cryoSPARC3.2.0final Euler assignment
12cryoSPARC3.2.0classification
13cryoSPARC3.2.03D reconstruction
20PHENIX1.19.2-4158-000model refinement
CTF correctionDetails: Patch CTF estimation (multi) from cryoSPARC. / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2037822
Details: Particle picking was performed using the template picker from cryoSPARC with templates generated from an ab initio model from the same dataset.
3D reconstructionResolution: 5.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 375961 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 303 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
Details: Helical 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.
Atomic model building
IDPDB-ID 3D fitting-ID
12D1B

2d1b

1
21

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