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- PDB-7v6c: Structure of the Dicer-2-R2D2 heterodimer bound to small RNA duplex -

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Basic information

Entry
Database: PDB / ID: 7v6c
TitleStructure of the Dicer-2-R2D2 heterodimer bound to small RNA duplex
Components
  • Dicer-2, isoform A
  • R2D2
  • RNA (5'-R(*AP*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*AP*G)-3')
  • RNA (5'-R(*CP*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*UP*C)-3')
  • RNA (5'-R(P*CP*CP*UP*CP*UP*CP*U)-3')
  • RNA (5'-R(P*UP*GP*AP*GP*G)-3')
KeywordsRNA BINDING PROTEIN / Ribonuclease / Double-stranded RNA-binding protein
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 / PKR-mediated signaling / dsRNA transport / regulation of regulatory ncRNA processing / dosage compensation by hyperactivation of X chromosome ...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 / PKR-mediated signaling / dsRNA transport / regulation of regulatory ncRNA processing / dosage compensation by hyperactivation of X chromosome / global gene silencing by mRNA cleavage / ribonuclease III / deoxyribonuclease I activity / apoptotic DNA fragmentation / detection of virus / RISC-loading complex / regulatory ncRNA-mediated post-transcriptional gene silencing / RISC complex assembly / ribonuclease III activity / 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 / locomotory behavior / mRNA 3'-UTR binding / helicase activity / cytoplasmic ribonucleoprotein granule / cellular response to virus / double-stranded RNA binding / defense response to virus / perinuclear region of cytoplasm / ATP hydrolysis activity / RNA binding / ATP binding / nucleus / 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 / LD06392p
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYamaguchi, S. / Nishizawa, T. / Kusakizako, T. / Yamashita, K. / Tomita, A. / Hirano, H. / Nishimasu, H. / Nureki, O.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)202111067 Japan
CitationJournal: Nature / Year: 2022
Title: Structure of the Dicer-2-R2D2 heterodimer bound to a small RNA duplex.
Authors: Sonomi Yamaguchi / Masahiro Naganuma / Tomohiro Nishizawa / Tsukasa Kusakizako / Yukihide Tomari / Hiroshi Nishimasu / Osamu Nureki /
Abstract: In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts. The RNase III enzyme Dicer-2 associates with its partner protein R2D2 ...In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts. The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation. Here we report cryo-electron microscopy structures of the Dicer-2-R2D2 and Dicer-2-R2D2-siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing.
History
DepositionAug 20, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 23, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Database references / Structure summary / Category: audit_author / citation_author
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
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dicer-2, isoform A
B: R2D2
E: RNA (5'-R(P*UP*GP*AP*GP*G)-3')
F: RNA (5'-R(P*CP*CP*UP*CP*UP*CP*U)-3')
C: RNA (5'-R(*AP*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*AP*G)-3')
D: RNA (5'-R(*CP*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*UP*C)-3')


Theoretical massNumber of molelcules
Total (without water)266,5186
Polymers266,5186
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10700 Å2
ΔGint-78 kcal/mol
Surface area86660 Å2

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 199309.000 Da / Num. of mol.: 1 / Mutation: M208L
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 R2D2 / R2D2 / R2d2 / isoform A / isoform B / isoform C


Mass: 39920.363 Da / Num. of mol.: 1
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

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RNA chain , 4 types, 4 molecules EFCD

#3: RNA chain RNA (5'-R(P*UP*GP*AP*GP*G)-3')


Mass: 6812.045 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#4: RNA chain RNA (5'-R(P*CP*CP*UP*CP*UP*CP*U)-3')


Mass: 6514.892 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#5: RNA chain RNA (5'-R(*AP*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*AP*G)-3')


Mass: 7141.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#6: RNA chain RNA (5'-R(*CP*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*UP*C)-3')


Mass: 6820.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) 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: Dicer-2-R2D2-siRNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144979 / Symmetry type: POINT
RefinementResolution: 3.3→165.75 Å / Cor.coef. Fo:Fc: 0.943 / SU B: 16.404 / SU ML: 0.268 / ESU R: 0.423
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.32422 --
obs0.32422 162512 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 101.99 Å2
Refinement stepCycle: 1 / Total: 15643
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01316120
ELECTRON MICROSCOPYr_bond_other_d0.0310.01714799
ELECTRON MICROSCOPYr_angle_refined_deg1.1851.62121966
ELECTRON MICROSCOPYr_angle_other_deg1.4491.61834131
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.4065.6792039
ELECTRON MICROSCOPYr_dihedral_angle_2_deg27.10522.475792
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.25152678
ELECTRON MICROSCOPYr_dihedral_angle_4_deg9.2591592
ELECTRON MICROSCOPYr_chiral_restr0.0870.2032164
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0217132
ELECTRON MICROSCOPYr_gen_planes_other0.0030.023686
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it7.1559.7387183
ELECTRON MICROSCOPYr_mcbond_other7.1559.7387182
ELECTRON MICROSCOPYr_mcangle_it11.85114.6088949
ELECTRON MICROSCOPYr_mcangle_other11.8514.6098950
ELECTRON MICROSCOPYr_scbond_it8.57512.1228937
ELECTRON MICROSCOPYr_scbond_other8.57512.128936
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other14.71517.83213017
ELECTRON MICROSCOPYr_long_range_B_refined26.30768198
ELECTRON MICROSCOPYr_long_range_B_other26.30768199
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.3→3.386 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork-11984 -
obs--100 %

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