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- EMDB-6904: Cryo-EM structure of human Dicer and its complexes with a pre-miR... -

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Basic information

Entry
Database: EMDB / ID: EMD-6904
TitleCryo-EM structure of human Dicer and its complexes with a pre-miRNA substrate
Map data
Sample
  • Complex: Dicer and trbp
    • Complex: Human Dicer and TRBP complex
      • Protein or peptide: Endoribonuclease Dicer
      • Protein or peptide: RISC-loading complex subunit TARBP2
Function / homology
Function and homology information


regulation of siRNA processing / regulation of miRNA processing / regulation of viral transcription / negative regulation of cytoplasmic pattern recognition receptor signaling pathway / regulation of regulatory ncRNA processing / peripheral nervous system myelin formation / negative regulation of defense response to virus by host / global gene silencing by mRNA cleavage / pre-miRNA binding / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis ...regulation of siRNA processing / regulation of miRNA processing / regulation of viral transcription / negative regulation of cytoplasmic pattern recognition receptor signaling pathway / regulation of regulatory ncRNA processing / peripheral nervous system myelin formation / negative regulation of defense response to virus by host / global gene silencing by mRNA cleavage / pre-miRNA binding / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / negative regulation of Schwann cell proliferation / tRNA decay / apoptotic DNA fragmentation / positive regulation of myelination / ribonuclease III / deoxyribonuclease I activity / positive regulation of Schwann cell differentiation / nerve development / RISC-loading complex / miRNA metabolic process / RISC complex assembly / ribonuclease III activity / miRNA processing / pre-miRNA processing / siRNA processing / siRNA binding / Regulation of MITF-M-dependent genes involved in apoptosis / M-decay: degradation of maternal mRNAs by maternally stored factors / pre-mRNA binding / RISC complex / miRNA binding / MicroRNA (miRNA) biogenesis / negative regulation of tumor necrosis factor production / negative regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of viral genome replication / protein sequestering activity / RNA endonuclease activity / neuron projection morphogenesis / helicase activity / negative regulation of protein kinase activity / PKR-mediated signaling / double-stranded RNA binding / regulation of translation / nuclear body / protein domain specific binding / negative regulation of gene expression / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / enzyme binding / protein homodimerization activity / RNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
RISC-loading complex subunit TRBP2 / TRBP2 , first double-stranded RNA binding domain / TRBP2 , second double-stranded RNA binding domain / TRBP2 , third double-stranded RNA binding domain / Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / : ...RISC-loading complex subunit TRBP2 / TRBP2 , first double-stranded RNA binding domain / TRBP2 , second double-stranded RNA binding domain / TRBP2 , third double-stranded RNA binding domain / Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / : / Dicer, platform domain / Dicer dimerisation 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 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
RISC-loading complex subunit TARBP2 / Endoribonuclease Dicer
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsLiu Z / Wang J / Cheng H / Ke X / Sun L / Zhang QC / Wang H-W
Funding support China, 1 items
OrganizationGrant numberCountry
National Science Foundation of China31530018 China
CitationJournal: Cell / Year: 2018
Title: Cryo-EM Structure of Human Dicer and Its Complexes with a Pre-miRNA Substrate.
Authors: Zhongmin Liu / Jia Wang / Hang Cheng / Xin Ke / Lei Sun / Qiangfeng Cliff Zhang / Hong-Wei Wang /
Abstract: Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse ...Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse range of double-stranded RNA (dsRNA) precursors to generate ∼22 nt microRNA (miRNA) or small interfering RNA (siRNA) products for sequence-directed gene silencing. In this work, we solved the cryoelectron microscopy (cryo-EM) structure of hDicer in complex with its cofactor protein TRBP and revealed the precise spatial arrangement of hDicer's multiple domains. We further solved structures of the hDicer-TRBP complex bound with pre-let-7 RNA in two distinct conformations. In combination with biochemical analysis, these structures reveal a property of the hDicer-TRBP complex to promote the stability of pre-miRNA's stem duplex in a pre-dicing state. These results provide insights into the mechanism of RNA processing by hDicer and illustrate the regulatory role of hDicer's N-terminal helicase domain.
History
DepositionFeb 7, 2018-
Header (metadata) releaseMay 9, 2018-
Map releaseMay 9, 2018-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5zak
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6904.png

Downloads & links

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Map

FileDownload / File: emd_6904.map.gz / Format: CCP4 / Size: 18.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 168 pix.
= 219.499 Å		1.31 Å/pix.
x 168 pix.
= 219.499 Å		1.31 Å/pix.
x 168 pix.
= 219.499 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.30654 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.047118355 - 0.116452485
Average (Standard dev.)0.0005593933 (±0.004350609)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 219.49872 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.30654166666671.30654166666671.3065416666667
M x/y/z168168168
origin x/y/z0.0000.0000.000
length x/y/z219.499219.499219.499
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS168168168
D min/max/mean-0.0470.1160.001

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Supplemental data

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Sample components

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Entire : Dicer and trbp

EntireName: Dicer and trbp
Components
  • Complex: Dicer and trbp
    • Complex: Human Dicer and TRBP complex
      • Protein or peptide: Endoribonuclease Dicer
      • Protein or peptide: RISC-loading complex subunit TARBP2

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Supramolecule #1: Dicer and trbp

SupramoleculeName: Dicer and trbp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 250 KDa

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Supramolecule #2: Human Dicer and TRBP complex

SupramoleculeName: Human Dicer and TRBP complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Endoribonuclease Dicer

MacromoleculeName: Endoribonuclease Dicer / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease III
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 218.947328 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKSPALQPLS MAGLQLMTPA SSPMGPFFGL PWQQEAIHDN IYTPRKYQVE LLEAALDHNT IVCLNTGSGK TFIAVLLTKE LSYQIRGDF SRNGKRTVFL VNSANQVAQQ VSAVRTHSDL KVGEYSNLEV NASWTKERWN QEFTKHQVLI MTCYVALNVL K NGYLSLSD ...String:
MKSPALQPLS MAGLQLMTPA SSPMGPFFGL PWQQEAIHDN IYTPRKYQVE LLEAALDHNT IVCLNTGSGK TFIAVLLTKE LSYQIRGDF SRNGKRTVFL VNSANQVAQQ VSAVRTHSDL KVGEYSNLEV NASWTKERWN QEFTKHQVLI MTCYVALNVL K NGYLSLSD INLLVFDECH LAILDHPYRE IMKLCENCPS CPRILGLTAS ILNGKCDPEE LEEKIQKLEK ILKSNAETAT DL VVLDRYT SQPCEIVVDC GPFTDRSGLY ERLLMELEEA LNFINDCNIS VHSKERDSTL ISKQILSDCR AVLVVLGPWC ADK VAGMMV RELQKYIKHE QEELHRKFLL FTDTFLRKIH ALCEEHFSPA SLDLKFVTPK VIKLLEILRK YKPYERQQFE SVEW YNNRN QDNYVSWSDS EDDDEDEEIE EKEKPETNFP SPFTNILCGI IFVERRYTAV VLNRLIKEAG KQDPELAYIS SNFIT GHGI GKNQPRNKQM EAEFRKQEEV LRKFRAHETN LLIATSIVEE GVDIPKCNLV VRFDLPTEYR SYVQSKGRAR APISNY IML ADTDKIKSFE EDLKTYKAIE KILRNKCSKS VDTGETDIDP VMDDDDVFPP YVLRPDDGGP RVTINTAIGH INRYCAR LP SDPFTHLAPK CRTRELPDGT FYSTLYLPIN SPLRASIVGP PMSCVRLAER VVALICCEKL HKIGELDDHL MPVGKETV K YEEELDLHDE EETSVPGRPG STKRRQCYPK AIPECLRDSY PRPDQPCYLY VIGMVLTTPL PDELNFRRRK LYPPEDTTR CFGILTAKPI PQIPHFPVYT RSGEVTISIE LKKSGFMLSL QMLELITRLH QYIFSHILRL EKPALEFKPT DADSAYCVLP LNVVNDSST LDIDFKFMED IEKSEARIGI PSTKYTKETP FVFKLEDYQD AVIIPRYRNF DQPHRFYVAD VYTDLTPLSK F PSPEYETF AEYYKTKYNL DLTNLNQPLL DVDHTSSRLN LLTPRHLNQK GKALPLSSAE KRKAKWESLQ NKQILVPELC AI HPIPASL WRKAVCLPSI LYRLHCLLTA EELRAQTASD AGVGVRSLPA DFRYPNLDFG WKKSIDSKSF ISISNSSSAE NDN YCKHST IVPENAAHQG ANRTSSLENH DQMSVNCRTL LSESPGKLHV EVSADLTAIN GLSYNQNLAN GSYDLANRDF CQGN QLNYY KQEIPVQPTT SYSIQNLYSY ENQPQPSDEC TLLSNKYLDG NANKSTSDGS PVMAVMPGTT DTIQVLKGRM DSEQS PSIG YSSRTLGPNP GLILQALTLS NASDGFNLER LEMLGDSFLK HAITTYLFCT YPDAHEGRLS YMRSKKVSNC NLYRLG KKK GLPSRMVVSI FDPPVNWLPP GYVVNQDKSN TDKWEKDEMT KDCMLANGKL DEDYEEEDEE EESLMWRAPK EEADYED DF LEYDQEHIRF IDNMLMGSGA FVKKISLSPF STTDSAYEWK MPKKSSLGSM PFSSDFEDFD YSSWDAMCYL DPSKAVEE D DFVVGFWNPS EENCGVDTGK QSISYDLHTE QCIADKSIAD CVEALLGCYL TSCGERAAQL FLCSLGLKVL PVIKRTDRE KALCPTRENF NSQQKNLSVS CAAASVASSR SSVLKDSEYG CLKIPPRCMF DHPDADKTLN HLISGFENFE KKINYRFKNK AYLLQAFTH ASYHYNTITD CYQRLEFLGD AILDYLITKH LYEDPRQHSP GVLTDLRSAL VNNTIFASLA VKYDYHKYFK A VSPELFHV IDDFVQFQLE KNEMQGMDSE LRRSEEDEEK EEDIEVPKAM GDIFESLAGA IYMDSGMSLE TVWQVYYPMM RP LIEKFSA NVPRSPVREL LEMEPETAKF SPAERTYDGK VRVTVEVVGK GKFKGVGRSY RIAKSAAARR ALRSLKANQP QVP NS

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Macromolecule #2: RISC-loading complex subunit TARBP2

MacromoleculeName: RISC-loading complex subunit TARBP2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.085277 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSEEEQGSGT TTGCGLPSIE QMLAANPGKT PISLLQEYGT RIGKTPVYDL LKAEGQAHQP NFTFRVTVGD TSCTGQGPSK KAAKHKAAE VALKHLKGGS MLEPALEDSS SFSPLDSSLP EDIPVFTAAA AATPVPSVVL TRSPPMELQP PVSPQQSECN P VGALQELV ...String:
MSEEEQGSGT TTGCGLPSIE QMLAANPGKT PISLLQEYGT RIGKTPVYDL LKAEGQAHQP NFTFRVTVGD TSCTGQGPSK KAAKHKAAE VALKHLKGGS MLEPALEDSS SFSPLDSSLP EDIPVFTAAA AATPVPSVVL TRSPPMELQP PVSPQQSECN P VGALQELV VQKGWRLPEY TVTQESGPAH RKEFTMTCRV ERFIEIGSGT SKKLAKRNAA AKMLLRVHTV PLDARDGNEV EP DDDHFSI GVGSRLDGLR NRGPGCTWDS LRNSVGEKIL SLRSCSLGSL GALGPACCRV LSELSEEQAF HVSYLDIEEL SLS GLCQCL VELSTQPATV CHGSATTREA ARGEAARRAL QYLKIMAGSK

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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: 8
Details: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 2 mM EDTA
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Details: The grid was glow-discharged prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-32 / Number grids imaged: 1 / Number real images: 7581 / Average exposure time: 8.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.5 µm / Calibrated defocus min: 2.5 µm / Calibrated magnification: 38462 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
Startup modelType of model: EMDB MAP
EMDB ID:

5601

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 337566
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 300000 / Software - Name: RELION (ver. 2.1)

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Atomic model buiding 1

Initial model
PDB IDChain

4gl2

chain_id: A, residue_range: 45-593

2kou

chain_id: A, residue_range: 624-726

5b16

chain_id: A, residue_range: 1286-1620

3c4t

chain_id: A, residue_range: 1653-1912
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 100 / Target criteria: CC
Output model

PDB-5zak:
Cryo-EM structure of human Dicer and its complexes with a pre-miRNA substrate

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