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- EMDB-27420: Structural Basis of MicroRNA Biogenesis by Dicer-1 and Its Partne... -

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Basic information

Entry
Database: EMDB / ID: EMD-27420
TitleStructural Basis of MicroRNA Biogenesis by Dicer-1 and Its Partner Protein Loqs-PB - complex IV
Map data
Sample
  • Complex: Ternary complex of Dicer-1 and Loqs-PB binding miRNA
    • Protein or peptide: Endoribonuclease Dcr-1
    • RNA: RNA (22-MER)
    • RNA: RNA (22-MER)
    • Protein or peptide: Loquacious, isoform BVerbosity
  • Ligand: URIDINE-5'-MONOPHOSPHATE
Function / homology
Function and homology information


mitotic cell cycle, embryonic / germarium-derived female germ-line cyst formation / MicroRNA (miRNA) biogenesis / germ-line stem cell division / germarium-derived oocyte fate determination / Small interfering RNA (siRNA) biogenesis / bidentate ribonuclease III activity / regulatory ncRNA processing / female germ-line stem cell asymmetric division / pole cell formation ...mitotic cell cycle, embryonic / germarium-derived female germ-line cyst formation / MicroRNA (miRNA) biogenesis / germ-line stem cell division / germarium-derived oocyte fate determination / Small interfering RNA (siRNA) biogenesis / bidentate ribonuclease III activity / regulatory ncRNA processing / female germ-line stem cell asymmetric division / pole cell formation / RISC complex binding / segment polarity determination / dosage compensation by hyperactivation of X chromosome / dsRNA transport / regulation of production of small RNA involved in gene silencing by RNA / pre-miRNA binding / germ-line stem cell population maintenance / apoptotic DNA fragmentation / deoxyribonuclease I activity / miRNA metabolic process / RISC-loading complex / RNA-mediated post-transcriptional gene silencing / RISC complex assembly / pre-miRNA processing / ribonuclease III activity / siRNA processing / miRNA processing / siRNA binding / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / RISC complex / dendrite morphogenesis / response to starvation / RNA phosphodiester bond hydrolysis / central nervous system development / double-stranded RNA binding / helicase activity / single-stranded RNA binding / RNA binding / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Staufen C-terminal domain / Staufen, C-terminal / Dicer, double-stranded RNA-binding domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family ...Staufen C-terminal domain / Staufen, C-terminal / Dicer, double-stranded RNA-binding domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / 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 / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Helicase, C-terminal / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Endoribonuclease Dcr-1 / Loquacious, isoform B
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsJouravleva K / Golovenko D / Demo G / Dutcher RC / Tanaka Hall TM / Zamore PD / Korostelev AA
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM136275 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM127094 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIA50165 United States
CitationJournal: Mol Cell / Year: 2022
Title: Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB.
Authors: Karina Jouravleva / Dmitrij Golovenko / Gabriel Demo / Robert C Dutcher / Traci M Tanaka Hall / Phillip D Zamore / Andrei A Korostelev /
Abstract: In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM ...In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs‑PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer‑1⋅Loqs‑PB heterodimer. The Dicer-1 dsRBD and three Loqs‑PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer‑1⋅Loqs‑PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
History
DepositionJun 23, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_27420.map.gz / Format: CCP4 / Size: 311.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 434 pix.
= 360.22 Å
0.83 Å/pix.
x 434 pix.
= 360.22 Å
0.83 Å/pix.
x 434 pix.
= 360.22 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.135
Minimum - Maximum-1.1159346 - 1.738469
Average (Standard dev.)3.4102304e-05 (±0.028414607)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions434434434
Spacing434434434
CellA=B=C: 360.22 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27420_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_27420_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

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Sample components

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Entire : Ternary complex of Dicer-1 and Loqs-PB binding miRNA

EntireName: Ternary complex of Dicer-1 and Loqs-PB binding miRNA
Components
  • Complex: Ternary complex of Dicer-1 and Loqs-PB binding miRNA
    • Protein or peptide: Endoribonuclease Dcr-1
    • RNA: RNA (22-MER)
    • RNA: RNA (22-MER)
    • Protein or peptide: Loquacious, isoform BVerbosity
  • Ligand: URIDINE-5'-MONOPHOSPHATE

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Supramolecule #1: Ternary complex of Dicer-1 and Loqs-PB binding miRNA

SupramoleculeName: Ternary complex of Dicer-1 and Loqs-PB binding miRNA / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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Macromolecule #1: Endoribonuclease Dcr-1

MacromoleculeName: Endoribonuclease Dcr-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 255.733797 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MAFHWCDNNL HTTVFTPRDF QVELLATAYE RNTIICLGHR SSKEFIALKL LQELSRRARR HGRVSVYLSC EVGTSTEPCS IYTMLTHLT DLRVWQEQPD MQIPFDHCWT DYHVSILRPE GFLYLLETRE LLLSRVELIV LEDCHDSAVY QRIRPLFENH I MPAPPADR ...String:
MAFHWCDNNL HTTVFTPRDF QVELLATAYE RNTIICLGHR SSKEFIALKL LQELSRRARR HGRVSVYLSC EVGTSTEPCS IYTMLTHLT DLRVWQEQPD MQIPFDHCWT DYHVSILRPE GFLYLLETRE LLLSRVELIV LEDCHDSAVY QRIRPLFENH I MPAPPADR PRILGLAGPL HSAGCELQQL SAMLATLEQS VLCQIESASD IVTVLRYCSR PHEYIVQCAP FEMDELSLVL AD VLNTHKS FLLDHRYDPY EIYGTDQFMD ELKDIPDPKV DPLNVINSLL VVLHEMGPWC TQRAAHHFYQ CNEKLKVKTP HER HYLLYC LVSTALIQLY SLCEHAFHRH LGSGSDSRQT IERYSSPKVR RLLQTLRCFK PEEVHTQADG LRRMRHQVDQ ADFN RLSHT LESKCRMVDQ LDQPPTETRA LVATLEQILH TTEDRQTNRS AARVTPTPTP AHAKPKPSSG ANTAQPRTRR RVYTR RHHR DHNDGSDTLC ALIYCNQNHT ARVLFELLAE ISRRDPDLKF LRCQYTTDRV ADPTTEPKEA ELEHRRQEEV LKRFRM HDC NVLIGTSVLE EGIDVPKCNL VVRWDPPTTY RSYVQCKGRA RAAPAYHVIL VAPSYKSPTV GSVQLTDRSH RYICATG DT TEADSDSDDS AMPNSSGSDP YTFGTARGTV KILNPEVFSK QPPTACDIKL QEIQDELPAS AQLDTSNSSD EAVSMSNT S PSESSTEQKS RRFQCELSSL TEPEDTSDTT AEIDTAHSLA STTKDLVHQM AQYREIEQML LSKCANTEPP EQEQCEAER FSACLAAYRP KPHLLTGASV DLGSAIALVN KYCARLPSDT FTKLTALWRC TRNERAGVTL FQYTLRLPIN SPLKHDIVGL PMPTQTLAR RLAALQACVE LHRIGELDDQ LQPIGKEGFR ALEPDWECFE LEPEDEQIVQ LSDEPRPGTT KRRQYYYKRI A SEFCDCRP VAGAPCYLYF IQLTLQCPIP EEQNTRGRKI YPPEDAQQGF GILTTKRIPK LSAFSIFTRS GEVKVSLELA KE RVILTSE QIVCINGFLN YTFTNVLRLQ KFLMLFDPDS TENCVFIVPT VKAPAGGKHI DWQFLELIQA NGNTMPRAVP DEE RQAQPF DPQRFQDAVV MPWYRNQDQP QYFYVAEICP HLSPLSCFPG DNYRTFKHYY LVKYGLTIQN TSQPLLDVDH TSAR LNFLT PRYVNRKGVA LPTSSEETKR AKRENLEQKQ ILVPELCTVH PFPASLWRTA VCLPCILYRI NGLLLADDIR KQVSA DLGL GRQQIEDEDF EWPMLDFGWS LSEVLKKSRE SKQKESLKDD TINGKDLVDV EKKAISEETQ IDKDSKDDKV EKSAIE LII EGEEKLQEAD DFIEIGTWSN DMADDIASFN QEDDDEDDAF HLPVLPANVK FCDQQTRYGS PTFWDVSNGE SGFKGPK SS QNKQGGKGKA KGPAKPTFNY YDSDNSLGSS YDDDDNAGPL NYMHHNYSSD DDDVADDIDA GRIAFTSKNE AETIETAQ E VEKRQKQLSI IQATNANERQ YQQTKNLLIG FNFKHEDQKE PATIRYEESI AKLKTEIESG GMLVPHDQQL VLKRSDAAE AQVAKVSMME LLKQLLPYVN EDVLAKKLGD RRELLLSDLV ELNADWVARH EQETYNVMGC GDSFDNYNDH HRLNLDEKQL KLQYERIEI EPPTSTKAIT SAILPAGFSF DRQPDLVGHP GPSPSIILQA LTMSNANDGI NLERLETIGD SFLKYAITTY L YITYENVH EGKLSHLRSK QVANLNLYRL GRRKRLGEYM IATKFEPHDN WLPPCYYVPK ELEKALIEAK IPTHHWKLAD LL DIKNLSS VQICEMVREK ADALGLEQNG GAQNGQLDDS NDSCNDFSCF IPYNLVSQHS IPDKSIADCV EALIGAYLIE CGP RGALLF MAWLGVRVLP ITRQLDGGNQ EQRIPGSTKP NAENVVTVYG AWPTPRSPLL HFAPNATEEL DQLLSGFEEF EESL GYKFR DRSYLLQAMT HASYTPNRLT DCYQRLEFLG DAVLDYLITR HLYEDPRQHS PGALTDLRSA LVNNTIFASL AVRHG FHKF FRHLSPGLND VIDRFVRIQQ ENGHCISEEY YLLSEEECDD AEDVEVPKAL GDVFESIAGA IFLDSNMSLD VVWHVY SNM MSPEIEQFSN SVPKSPIREL LELEPETAKF GKPEKLADGR RVRVTVDVFC KGTFRGIGRN YRIAKCTAAK CALRQLK KQ GLIAKKD

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Macromolecule #4: Loquacious, isoform B

MacromoleculeName: Loquacious, isoform B / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 50.149867 KDa
Recombinant expressionOrganism: unidentified baculovirus
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 EPRSSENYYG ELKDISVPTL TTQHSNKVSQ FHKTLKNATG KKLLKLQKTC LKNNKIDYIK LLGE IATEN QFEVTYVDIE EKTFSGQFQC LVQLSTLPVG VCHGSGPTAA DAQRHAAQNA LEYLKIMTKK

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Macromolecule #2: RNA (22-MER)

MacromoleculeName: RNA (22-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 6.835085 KDa
SequenceString:
GAGGUAGUAG GUUGUAUAGU A

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Macromolecule #3: RNA (22-MER)

MacromoleculeName: RNA (22-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 6.820074 KDa
SequenceString:
CUAUACAACC UACUACCUCU CU

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Macromolecule #5: URIDINE-5'-MONOPHOSPHATE

MacromoleculeName: URIDINE-5'-MONOPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: U5P
Molecular weightTheoretical: 324.181 Da
Chemical component information

ChemComp-U:
URIDINE-5'-MONOPHOSPHATE / Uridine monophosphate

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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

Concentration1 mg/mL
BufferpH: 7.9 / Component - Concentration: 20.0 mM / Component - Formula: HEPES-KOH / Component - Name: HEPES-KOH
GridModel: C-flat-1.2/1.3 / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3161540
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final 3D classificationNumber classes: 6 / Software - Name: RELION
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 120261

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