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- PDB-7w0e: dmDicer2-LoqsPD-dsRNA Active-dicing status -

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Basic information

Entry
Database: PDB / ID: 7w0e
TitledmDicer2-LoqsPD-dsRNA Active-dicing status
Components
  • Dicer-2, isoform A
  • Loquacious, isoform D
  • dsRNA
KeywordsRNA BINDING PROTEIN / Ribonuclease
Function / homology
Function and homology information


positive regulation of Toll signaling pathway / lncRNA catabolic process / MicroRNA (miRNA) biogenesis / RNAi-mediated antiviral immune response / Small interfering RNA (siRNA) biogenesis / PKR-mediated signaling / female germ-line stem cell asymmetric division / regulation of regulatory ncRNA processing / dosage compensation by hyperactivation of X chromosome / RISC complex binding ...positive regulation of Toll signaling pathway / lncRNA catabolic process / MicroRNA (miRNA) biogenesis / RNAi-mediated antiviral immune response / Small interfering RNA (siRNA) biogenesis / PKR-mediated signaling / female germ-line stem cell asymmetric division / regulation of regulatory ncRNA processing / dosage compensation by hyperactivation of X chromosome / RISC complex binding / global gene silencing by mRNA cleavage / apoptotic DNA fragmentation / germ-line stem cell population maintenance / ribonuclease III / deoxyribonuclease I activity / detection of virus / RISC-loading complex / miRNA metabolic process / 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 / RISC complex / positive regulation of innate immune response / positive regulation of defense response to virus by host / central nervous system development / mRNA 3'-UTR binding / locomotory behavior / helicase activity / cellular response to virus / cytoplasmic ribonucleoprotein granule / heterochromatin formation / 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 superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family ...: / : / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / 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 profile. / PAZ domain / 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 domain / DEAD/DEAH box helicase / 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
ADENOSINE-5'-DIPHOSPHATE / RNA / RNA (> 10) / Endoribonuclease Dcr-2 / Protein Loquacious
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsSu, S. / Wang, J. / Wang, H.W. / Ma, J.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)2017YFA0503500 China
National Natural Science Foundation of China (NSFC)32000849 China
CitationJournal: Nature / Year: 2022
Title: Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD.
Authors: Shichen Su / Jia Wang / Ting Deng / Xun Yuan / Jinqiu He / Nan Liu / Xiaomin Li / Ying Huang / Hong-Wei Wang / Jinbiao Ma /
Abstract: Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided ...Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs). ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes. Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD.
History
DepositionNov 18, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 27, 2022Provider: repository / Type: Initial release
Revision 1.1May 4, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.year
Revision 1.2Jul 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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
Revision 1.3Jul 27, 2022Group: 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
Revision 1.4Jun 26, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: dsRNA
D: dsRNA
A: Dicer-2, isoform A
B: Loquacious, isoform D
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,9628
Polymers270,4614
Non-polymers5004
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain dsRNA


Mass: 16976.076 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#2: Protein Dicer-2, isoform A / FI15132p1


Mass: 198006.688 Da / Num. of mol.: 1
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
#3: Protein Loquacious, isoform D


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: Escherichia coli (E. coli) / References: UniProt: M9MRT5
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestN

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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: Dicer2-LoqsPD-dsRNA complex at its active-dicing state
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental 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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 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: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57679 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 96.07 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315376
ELECTRON MICROSCOPYf_angle_d0.57121303
ELECTRON MICROSCOPYf_dihedral_angle_d7.8042947
ELECTRON MICROSCOPYf_chiral_restr0.042477
ELECTRON MICROSCOPYf_plane_restr0.0032338

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