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- EMDB-34709: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state -

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Basic information

Entry
Database: EMDB / ID: EMD-34709
TitleDmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state
Map data
Sample
  • Complex: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state
KeywordsRibonuclease / DOUBLE STRANDED RNA / RNA BINDING PROTEIN-RNA COMPLEX
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.74 Å
AuthorsSu S / Wang J / Wang HW / Ma J
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32000849 China
CitationJournal: Nat Commun / Year: 2023
Title: Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.
Authors: Ting Deng / Shichen Su / Xun Yuan / Jinqiu He / Ying Huang / Jinbiao Ma / Jia Wang /
Abstract: Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated ...Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.
History
DepositionNov 9, 2022-
Header (metadata) releaseSep 6, 2023-
Map releaseSep 6, 2023-
UpdateSep 6, 2023-
Current statusSep 6, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_34709.png

Downloads & links

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Map

FileDownload / File: emd_34709.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0742 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.49887326 - 1.1317103
Average (Standard dev.)0.004792429 (±0.039525073)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 343.74402 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_34709_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34709_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state

EntireName: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state
Components
  • Complex: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state

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Supramolecule #1: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state

SupramoleculeName: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Trimer state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 300 KDa

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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

Concentration0.3 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 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: 1.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 77693
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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