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- EMDB-19759: Small circular RNA dimer - Class 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-19759
TitleSmall circular RNA dimer - Class 3
Map dataDimer map C2 symmetry
Sample
  • Complex: Dimer composed of two small circular RNAs and two complimentary RNAs
    • RNA: RNA (36-MER)
    • RNA: RNA (36-MER)
Keywordssmall RNA circles / dimer / RNA crossover / RNA
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsMcRae EK / Kristoffersen EL / Holliger P / Andersen ES
Funding support Denmark, 1 items
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
DepositionFeb 28, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19759.png

Downloads & links

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Map

FileDownload / File: emd_19759.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDimer map C2 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.29 Å/pix.
x 216 pix.
= 279.504 Å		1.29 Å/pix.
x 216 pix.
= 279.504 Å		1.29 Å/pix.
x 216 pix.
= 279.504 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: 0.2
Minimum - Maximum-0.22866164 - 0.58293086
Average (Standard dev.)0.00020265997 (±0.014107473)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 279.504 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_19759_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_19759_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimer composed of two small circular RNAs and two complimentary RNAs

EntireName: Dimer composed of two small circular RNAs and two complimentary RNAs
Components
  • Complex: Dimer composed of two small circular RNAs and two complimentary RNAs
    • RNA: RNA (36-MER)
    • RNA: RNA (36-MER)

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Supramolecule #1: Dimer composed of two small circular RNAs and two complimentary RNAs

SupramoleculeName: Dimer composed of two small circular RNAs and two complimentary RNAs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Dimer prepared by annealing
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: RNA (36-MER)

MacromoleculeName: RNA (36-MER) / type: rna / ID: 1 / Number of copies: 2
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.817235 KDa
SequenceString:
UGGAAGAAAU CGAAGAAGAU GAAGAACGCG AAGAAC

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Macromolecule #2: RNA (36-MER)

MacromoleculeName: RNA (36-MER) / type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.191466 KDa
SequenceString:
AUCUUCUUCG AUUUCUUCCA GUUCUUCGCG UUCUUC

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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

BufferpH: 7.5
GridMaterial: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 10 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS 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: 2 / Number real images: 6820 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
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 defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / 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

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 116769
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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