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- PDB-8hf0: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Dimer state -

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Basic information

Entry
Database: PDB / ID: 8hf0
TitleDmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Dimer state
Components
  • Dicer-2, isoform A
  • LD06392p
  • Loquacious, isoform DVerbosity
  • RNA (52-MER)
KeywordsRNA BINDING PROTEIN/RNA / Ribonuclease / DOUBLE STRANDED RNA / RNA BINDING PROTEIN-RNA COMPLEX
Function / homology
Function and homology information


follicle cell of egg chamber stalk formation / regulatory ncRNA processing / bidentate ribonuclease III activity / positive regulation of Toll signaling pathway / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / female germ-line stem cell asymmetric division / PKR-mediated signaling / regulation of regulatory ncRNA processing / dsRNA transport ...follicle cell of egg chamber stalk formation / regulatory ncRNA processing / bidentate ribonuclease III activity / positive regulation of Toll signaling pathway / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / female germ-line stem cell asymmetric division / PKR-mediated signaling / regulation of regulatory ncRNA processing / dsRNA transport / dosage compensation by hyperactivation of X chromosome / RISC complex binding / global gene silencing by mRNA cleavage / germ-line stem cell population maintenance / ribonuclease III / apoptotic DNA fragmentation / miRNA metabolic process / deoxyribonuclease I activity / RISC-loading complex / detection of virus / RISC complex assembly / regulatory ncRNA-mediated post-transcriptional gene silencing / ribonuclease III activity / pre-miRNA processing / siRNA processing / siRNA binding / ATP-dependent activity, acting on RNA / positive regulation of innate immune response / RISC complex / heterochromatin formation / positive regulation of defense response to virus by host / helicase activity / locomotory behavior / central nervous system development / mRNA 3'-UTR binding / cellular response to virus / cytoplasmic ribonucleoprotein granule / double-stranded RNA binding / defense response to virus / perinuclear region of cytoplasm / ATP hydrolysis activity / RNA binding / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain ...: / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ / PAZ domain / PAZ domain / PAZ domain profile. / Double-stranded RNA binding motif / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / Endoribonuclease Dcr-2 / Protein Loquacious / LD06392p
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsSu, S. / Wang, J. / Wang, H.W. / Ma, J.
Funding support China, 1items
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, 2022Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dicer-2, isoform A
B: Loquacious, isoform D
C: LD06392p
D: Dicer-2, isoform A
E: LD06392p
P: RNA (52-MER)
N: RNA (52-MER)


Theoretical massNumber of molelcules
Total (without water)538,0407
Polymers538,0407
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Dicer-2, isoform A / / FI15132p1


Mass: 198006.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Dcr-2, cg6493, Dcr, dcr, DCR-2, dcr-2, Dcr-2-RA, DCR2, Dcr2, dcr2, dDcr2, dic2, DICER, Dicer, dicer, DICER-2, dicer-2, Dicer2, dicer2, dmDcr-2, Dmel\CG6493, CG6493, Dmel_CG6493
Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: A1ZAW0, deoxyribonuclease I, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters, ribonuclease III, EC: 3.6.1.3
#2: Protein Loquacious, isoform D / Verbosity


Mass: 38502.574 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: loqs, cg6866, Dmel\CG6866, dRax, loq, LOQS, Loqs, Loqs-PD, LqPD, R3D1, r3d1, R3D1-L, R3D1-S, TRBP, CG6866, Dmel_CG6866
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: M9MRT5
#3: Protein LD06392p / R2D2 / R2d2 / isoform A / isoform B / isoform C


Mass: 35115.254 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: r2d2, cg7138, Dmel\CG7138, R2D2, R2d2, CG7138, Dmel_CG7138
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9VLW8
#4: RNA chain RNA (52-MER)


Mass: 16646.871 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: dsRNA / Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Production host: Drosophila melanogaster (fruit fly)

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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: DmDcr-2/R2D2/LoqsPD with 50bp-dsRNA in Dimer state / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 defocus max: 1500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141544 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00330334
ELECTRON MICROSCOPYf_angle_d0.56241488
ELECTRON MICROSCOPYf_dihedral_angle_d8.8234846
ELECTRON MICROSCOPYf_chiral_restr0.044733
ELECTRON MICROSCOPYf_plane_restr0.0044953

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