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- PDB-9h86: Small circular RNA dimer - Class 4 -

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Basic information

Entry
Database: PDB / ID: 9h86
TitleSmall circular RNA dimer - Class 4
Components
  • Circular RNA
  • Complementary strand
KeywordsRNA / small RNA circles / dimer / RNA crossover
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.3 Å
AuthorsMcRae, E.K. / Kristoffersen, E.L. / Holliger, P. / Andersen, E.S.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF21OC0070452 Denmark
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Roles of dimeric intermediates in RNA-catalyzed rolling circle synthesis.
Authors: Emil L Kristoffersen / Ewan K S McRae / Niels R Sørensen / Philipp Holliger / Ebbe S Andersen /
Abstract: The RNA world hypothesis is supported by the discovery of RNA polymerase ribozymes that can perform RNA-catalyzed RNA replication processes on different RNA templates. Recently, RNA-catalyzed rolling ...The RNA world hypothesis is supported by the discovery of RNA polymerase ribozymes that can perform RNA-catalyzed RNA replication processes on different RNA templates. Recently, RNA-catalyzed rolling circle synthesis (RCS) on small circular RNA (scRNA) templates has been demonstrated. However, the structural and dynamic properties of scRNA replication and its products and intermediates have not been explored. Here, we have used cryogenic electron microscopy (cryo-EM) to characterize products and intermediates relevant for RCS replication. We find that these form an unexpectedly diverse group of RNA nanostructures. The main structural motif observed is a fully hybridized dimeric complex composed of two scRNAs and their complement strands resolved to 5.3 Å. Cryo-EM also reveals higher-order dimer filaments and dimer assembly intermediates, suggesting an assembly mechanism for the observed complexes. We show that the dimer complexes are stable and inhibit RNA-catalyzed RCS but can be reactivated by addition of more scRNA templates. We propose dimer formation as a general property of RCS replication and speculate that dimers might have benefited a primordial RNA genetic system by providing a stable ''storage'' form for RNA replication products and by coordinated RNA replication on both scRNA template strands.
History
DepositionOct 28, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 18, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_DOI / _citation.title ..._citation.pdbx_database_id_DOI / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Jun 25, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Complementary strand
D: Circular RNA
C: Circular RNA
B: Complementary strand
E: Complementary strand
H: Circular RNA
G: Circular RNA
F: Complementary strand


Theoretical massNumber of molelcules
Total (without water)92,0358
Polymers92,0358
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chain
Complementary strand


Mass: 11817.235 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#2: RNA chain
Circular RNA


Mass: 11191.466 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Circular RNA / Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
Has protein modificationN

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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: Dimer intermediate class 2 composed of 2 circular RNA and 2 complimentary RNA
Type: COMPLEX / Details: Dimer prepared by annealing / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 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: 2 / Num. of real images: 6820
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.5.3particle selection
2EPUimage acquisition
4cryoSPARC4.5.3CTF correction
7UCSF ChimeraX1.6.1model fitting
11cryoSPARC4.5.3classification
12cryoSPARC4.5.33D reconstructionNU-refinement
13PHENIX1.21.2-5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 668921
3D reconstructionResolution: 9.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26859 / Symmetry type: POINT
Atomic model buildingB value: 231 / Protocol: OTHER / Space: REAL
RefinementCross valid method: NONE

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