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- EMDB-34708: DmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state -

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Basic information

Entry
Database: EMDB / ID: EMD-34708
TitleDmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state
Map data
Sample
  • Complex: DmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state
    • Protein or peptide: Dicer-2, isoform A
    • Protein or peptide: Loquacious, isoform D
    • Protein or peptide: LD06392p
    • RNA: RNA (5'-R(P*GP*AP*GP*AP*CP*UP*UP*GP*G)-3')
    • RNA: RNA (5'-R(P*CP*CP*AP*AP*GP*UP*CP*UP*CP*UP*U)-3')
KeywordsRibonuclease / DOUBLE STRANDED RNA / RNA BINDING PROTEIN-RNA COMPLEX
Function / homology
Function and homology information


follicle cell of egg chamber stalk formation / lncRNA catabolic process / : / positive regulation of Toll signaling pathway / regulatory ncRNA processing / MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / female germ-line stem cell asymmetric division / PKR-mediated signaling / regulation of regulatory ncRNA processing ...follicle cell of egg chamber stalk formation / lncRNA catabolic process / : / positive regulation of Toll signaling pathway / regulatory ncRNA processing / 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 / RISC-loading complex / miRNA metabolic process / deoxyribonuclease I activity / detection of virus / RISC complex assembly / ribonuclease III activity / regulatory ncRNA-mediated post-transcriptional gene silencing / siRNA binding / pre-miRNA processing / ATP-dependent activity, acting on RNA / siRNA processing / 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 / cytoplasm / cytosol
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 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 / 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
Endoribonuclease Dcr-2 / Protein Loquacious / LD06392p
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSu S / Wang J / 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_34708.png

Downloads & links

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Map

FileDownload / File: emd_34708.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.4515323 - 1.0811157
Average (Standard dev.)0.003017904 (±0.05322392)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34708_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34708_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 19bp-dsRNA in Trimer state

EntireName: DmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state
Components
  • Complex: DmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state
    • Protein or peptide: Dicer-2, isoform A
    • Protein or peptide: Loquacious, isoform D
    • Protein or peptide: LD06392p
    • RNA: RNA (5'-R(P*GP*AP*GP*AP*CP*UP*UP*GP*G)-3')
    • RNA: RNA (5'-R(P*CP*CP*AP*AP*GP*UP*CP*UP*CP*UP*U)-3')

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Supramolecule #1: DmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state

SupramoleculeName: DmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Dicer-2, isoform A

MacromoleculeName: Dicer-2, isoform A / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: deoxyribonuclease I
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 197.875484 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EDVEIKPRGY QLRLVDHLTK SNGIVYLPTG SGKTFVAILV LKRFSQDFDK PIESGGKRAL FMCNTVELAR QQAMAVRRCT NFKVGFYVG EQGVDDWTRG MWSDEIKKNQ VLVGTAQVFL DMVTQTYVAL SSLSVVIIDE CHHGTGHHPF REFMRLFTIA N QTKLPRVV ...String:
EDVEIKPRGY QLRLVDHLTK SNGIVYLPTG SGKTFVAILV LKRFSQDFDK PIESGGKRAL FMCNTVELAR QQAMAVRRCT NFKVGFYVG EQGVDDWTRG MWSDEIKKNQ VLVGTAQVFL DMVTQTYVAL SSLSVVIIDE CHHGTGHHPF REFMRLFTIA N QTKLPRVV GLTGVLIKGN EITNVATKLK ELEITYRGNI ITVSDTKEME NVMLYATKPT EVMVSFPHQE QVLTVTRLIS AE IEKFYVS LDLMNIGVQP IRRSKSLQCL RDPSKKSFVK QLFNDFLYQM KEYGIYAASI AIISLIVEFD IKRRQAETLS VKL MHRTAL TLCEKIRHLL VQKLQDMTYD DDDDNVNTEE VIMNFSTPKV QRFLMSLKVS FADKDPKDIC CLVFVERRYT CKCI YGLLL NYIQSTPELR NVLTPQFMVG RNNISPDFES VLERKWQKSA IQQFRDGNAN LMICSSVLEE GIDVQACNHV FILDP VKTF NMYVQSKGRA RTTEAKFVLF TADKEREKTI QQIYQYRKAH NDIAEYLKDR VLEKTEPELY EIKGHFQDDI DPFTNE NGA VLLPNNALAI LHRYCQTIPT DAFGFVIPWF HVLQEDERDR IFGVSAKGKH VISINMPVNC MLRDTIYSDP MDNVKTA KI SAAFKACKVL YSLGELNERF VPKTLKERVA SIADVHFEHW NKYGDSVTAT VNKADKSKDR TYKTECPLEF YDALPRVG E ICYAYEIFLE PQFESCEYTE HMYLNLQTPR NYAILLRNKL PRLAEMPLFS NQGKLHVRVA NAPLEVIIQN SEQLELLHQ FHGMVFRDIL KIWHPFFVLD RRSKENSYLV VPLILGAGEQ KCFDWELMTN FRRLPQSHGS NVQQREQQPA PRPEDFEGKI VTQWYANYD KPMLVTKVHR ELTPLSYMEK NQQDKTYYEF TMSKYGNRIG DVVHKDKFMI EVRDLTEQLT FYVHNRGKFN A KSKAKMKV ILIPELCFNF NFPGDLWLKL IFLPSILNRM YFLLHAEALR KRFNTYLNLH LLPFNGTDYM PRPLEIDYSL KR NVDPLGN VIPTEDIEEP KSLLEPMPTK SIEASVANLE ITEFENPWQK YMEPVDLSRN LLSTYPVELD YYYHFSVGNV CEM NEMDFE DKEYWAKNQF HMPTGNIYGN RTPAKTNANV PALMPSKPTV RGKVKPLLIL QKTVSKEHIT PAEQGEFLAA ITAS SAADV FDMERLEILG NSFLKLSATL YLASKYSDWN EGTLTEVKSK LVSNRNLLFC LIDADIPKTL NTIQFTPRYT WLPPG ISLP HNVLALWREN PEFAKIIGPH NLRDLALGDE ESLVKGNCSD INYNRFVEGC RANGQSFYAG ADFSSEVNFC VGLVTI PNK VIADTLEALL GVIVKNYGLQ HAFKMLEYFK ICRADIDKPL TQLLNLELGG KKMRANVNTT EIDGFLINHY YLEKNLG YT FKDRRYLLQA LTHPSYPTNR ITGSYQELEF IGNAILDFLI SAYIFENNTK MNPGALTDLR SALVNNTTLA CICVRHRL H FFILAENAKL SEIISKFVNF QESQGHRVTN YVRILLEEAD VQPTPLDLDD ELDMTELPHA NKCISQEAEK GVPPKGEFN MSTNVDVPKA LGDVLEALIA AVYLDCRDLQ RTWEVIFNLF EPELQEFTRK VPINHIRQLV EHKHAKPVFS SPIVEGETVM VSCQFTCME KTIKVYGFGS NKDQAKLSAA KHALQQLSKC DA

UniProtKB: Endoribonuclease Dcr-2

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Macromolecule #2: Loquacious, isoform D

MacromoleculeName: Loquacious, isoform D / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 2.033304 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
IDRYEQVSKD FEFIKI

UniProtKB: Protein Loquacious

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Macromolecule #3: LD06392p

MacromoleculeName: LD06392p / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 14.706812 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MEESMEELEA LRRKKFTTYW ELKEAGSVDH TGMRLCDRHN YFKNFYPTLK KEAIEAINSD EYESSKDKAM DVMSSLKITP KISEVESSS LVPLLSVELN CAFDVVLMAK ETDIYDHIID YFRTMLI

UniProtKB: LD06392p

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Macromolecule #4: RNA (5'-R(P*GP*AP*GP*AP*CP*UP*UP*GP*G)-3')

MacromoleculeName: RNA (5'-R(P*GP*AP*GP*AP*CP*UP*UP*GP*G)-3') / type: rna / ID: 4 / Number of copies: 3
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 2.911791 KDa
SequenceString:
GAGACUUGG

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Macromolecule #5: RNA (5'-R(P*CP*CP*AP*AP*GP*UP*CP*UP*CP*UP*U)-3')

MacromoleculeName: RNA (5'-R(P*CP*CP*AP*AP*GP*UP*CP*UP*CP*UP*U)-3') / type: rna / ID: 5 / Number of copies: 3
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 3.40405 KDa
SequenceString:
CCAAGUCUCU U

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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: 0.01 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) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 46899
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8hf1:
DmDcr-2/R2D2/LoqsPD with 19bp-dsRNA in Trimer state

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