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- EMDB-32241: dmDicer2-LoqsPD-dsRNA Post-dicing status -

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Basic information

Entry
Database: EMDB / ID: EMD-32241
TitledmDicer2-LoqsPD-dsRNA Post-dicing status
Map data
Sample
  • Complex: Dicer2-LoqsPD-dsRNA complex at its post-dicing state
    • Protein or peptide: Dicer-2, isoform A
    • Protein or peptide: Loquacious, isoform DVerbosity
    • RNA: siRNA
Function / homology
Function and homology information


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 / dsRNA transport / regulation of regulatory ncRNA processing / dosage compensation by hyperactivation of X chromosome ...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 / dsRNA transport / regulation of regulatory ncRNA processing / dosage compensation by hyperactivation of X chromosome / RISC complex binding / global gene silencing by mRNA cleavage / germ-line stem cell population maintenance / ribonuclease III / deoxyribonuclease I activity / apoptotic DNA fragmentation / miRNA metabolic process / RISC-loading complex / regulatory ncRNA-mediated post-transcriptional gene silencing / detection of virus / RISC complex assembly / pre-miRNA processing / 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 / central nervous system development / 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 / 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
Endoribonuclease Dcr-2 / Protein Loquacious
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.55 Å
AuthorsSu S / Wang J / Wang HW / Ma J
Funding support China, 2 items
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, 2021-
Header (metadata) releaseApr 27, 2022-
Map releaseApr 27, 2022-
UpdateJul 27, 2022-
Current statusJul 27, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32241.png

Downloads & links

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Map

FileDownload / File: emd_32241.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.26906583 - 0.8159921
Average (Standard dev.)0.0010577403 (±0.034126136)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Dicer2-LoqsPD-dsRNA complex at its post-dicing state

EntireName: Dicer2-LoqsPD-dsRNA complex at its post-dicing state
Components
  • Complex: Dicer2-LoqsPD-dsRNA complex at its post-dicing state
    • Protein or peptide: Dicer-2, isoform A
    • Protein or peptide: Loquacious, isoform DVerbosity
    • RNA: siRNA

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Supramolecule #1: Dicer2-LoqsPD-dsRNA complex at its post-dicing state

SupramoleculeName: Dicer2-LoqsPD-dsRNA complex at its post-dicing state / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Dicer-2, isoform A

MacromoleculeName: Dicer-2, isoform A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: deoxyribonuclease I
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 198.008656 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEDVEIKPRG YQLRLVDHLT KSNGIVYLPT GSGKTFVAIL VLKRFSQDFD KPIESGGKRA LFMCNTVELA RQQAMAVRRC TNFKVGFYV GEQGVDDWTR GMWSDEIKKN QVLVGTAQVF LDMVTQTYVA LSSLSVVIID ECHHGTGHHP FREFMRLFTI A NQTKLPRV ...String:
MEDVEIKPRG YQLRLVDHLT KSNGIVYLPT GSGKTFVAIL VLKRFSQDFD KPIESGGKRA LFMCNTVELA RQQAMAVRRC TNFKVGFYV GEQGVDDWTR GMWSDEIKKN QVLVGTAQVF LDMVTQTYVA LSSLSVVIID ECHHGTGHHP FREFMRLFTI A NQTKLPRV VGLTGVLIKG NEITNVATKL KELEITYRGN IITVSDTKEM ENVMLYATKP TEVMVSFPHQ EQVLTVTRLI SA EIEKFYV SLDLMNIGVQ PIRRSKSLQC LRDPSKKSFV KQLFNDFLYQ MKEYGIYAAS IAIISLIVEF DIKRRQAETL SVK LMHRTA LTLCEKIRHL LVQKLQDMTY DDDDDNVNTE EVIMNFSTPK VQRFLMSLKV SFADKDPKDI CCLVFVERRY TCKC IYGLL LNYIQSTPEL RNVLTPQFMV GRNNISPDFE SVLERKWQKS AIQQFRDGNA NLMICSSVLE EGIDVQACNH VFILD PVKT FNMYVQSKGR ARTTEAKFVL FTADKEREKT IQQIYQYRKA HNDIAEYLKD RVLEKTEPEL YEIKGHFQDD IDPFTN ENG AVLLPNNALA ILHRYCQTIP TDAFGFVIPW FHVLQEDERD RIFGVSAKGK HVISINMPVN CMLRDTIYSD PMDNVKT AK ISAAFKACKV LYSLGELNER FVPKTLKERV ASIADVHFEH WNKYGDSVTA TVNKADKSKD RTYKTECPLE FYDALPRV G EICYAYEIFL EPQFESCEYT EHMYLNLQTP RNYAILLRNK LPRLAEMPLF SNQGKLHVRV ANAPLEVIIQ NSEQLELLH QFHGMVFRDI LKIWHPFFVL DRRSKENSYL VVPLILGAGE QKCFDWELMT NFRRLPQSHG SNVQQREQQP APRPEDFEGK IVTQWYANY DKPMLVTKVH RELTPLSYME KNQQDKTYYE FTMSKYGNRI GDVVHKDKFM IEVRDLTEQL TFYVHNRGKF N AKSKAKMK VILIPELCFN FNFPGDLWLK LIFLPSILNR MYFLLHAEAL RKRFNTYLNL HLLPFNGTDY MPRPLEIDYS LK RNVDPLG NVIPTEDIEE PKSLLEPMPT KSIEASVANL EITEFENPWQ KYMEPVDLSR NLLSTYPVEL DYYYHFSVGN VCE MNEMDF EDKEYWAKNQ FHMPTGNIYG NRTPAKTNAN VPALMPSKPT VRGKVKPLLI LQKTVSKEHI TPAEQGEFLA AITA SSAAD VFDMERLEIL GDSFLKLSAT LYLASKYSDW NEGTLTEVKS KLVSNRNLLF CLIDADIPKT LNTIQFTPRY TWLPP GISL PHNVLALWRE NPEFAKIIGP HNLRDLALGD EESLVKGNCS DINYNRFVEG CRANGQSFYA GADFSSEVNF CVGLVT IPN KVIADTLEAL LGVIVKNYGL QHAFKMLEYF KICRADIDKP LTQLLNLELG GKKMRANVNT TEIDGFLINH YYLEKNL GY TFKDRRYLLQ ALTHPSYPTN RITGSYQELE FIGDAILDFL ISAYIFENNT KMNPGALTDL RSALVNNTTL ACICVRHR L HFFILAENAK LSEIISKFVN FQESQGHRVT NYVRILLEEA DVQPTPLDLD DELDMTELPH ANKCISQEAE KGVPPKGEF NMSTNVDVPK ALGDVLEALI AAVYLDCRDL QRTWEVIFNL FEPELQEFTR KVPINHIRQL VEHKHAKPVF SSPIVEGETV MVSCQFTCM EKTIKVYGFG SNKDQAKLSA AKHALQQLSK CDA

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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: 38.502574 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDQENFHGSS LPQQLQNLHI QPQQASPNPV QTGFAPRRHY NNLVGLGNGN AVSGSPVKGA PLGQRHVKLK KEKISAQVAQ LSQPGQLQL SDVGDPALAG GSGLQGGVGL MGVILPSDEA LKFVSETDAN GLAMKTPVSI LQELLSRRGI TPGYELVQIE G AIHEPTFR ...String:
MDQENFHGSS LPQQLQNLHI QPQQASPNPV QTGFAPRRHY NNLVGLGNGN AVSGSPVKGA PLGQRHVKLK KEKISAQVAQ LSQPGQLQL SDVGDPALAG GSGLQGGVGL MGVILPSDEA LKFVSETDAN GLAMKTPVSI LQELLSRRGI TPGYELVQIE G AIHEPTFR FRVSFKDKDT PFTAMGAGRS KKEAKHAAAR ALIDKLIGAQ LPESPSSSAG PSVTGLTVAG SGGDGNANAT GG GDASDKT VGNPIGWLQE MCMQRRWPPP SYETETEVGL PHERLFTIAC SILNYREMGK GKSKKIAKRL AAHRMWMRLQ ETP IDSGKI SDSICGELEG EVSIIQDIDR YEQVSKDFEF IKI

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Macromolecule #3: siRNA

MacromoleculeName: siRNA / type: rna / ID: 3 / Number of copies: 4
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 16.646871 KDa
SequenceString:
GAGACUUGGG CAAUGUGACU GCUGAUCAGC AGUCACAUUG CCCAAGUCUC UU

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 82469

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7w0f:
dmDicer2-LoqsPD-dsRNA Post-dicing status

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