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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-5605 | |||||||||
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Title | Substrate-specific structural rearrangements of human Dicer | |||||||||
![]() | Negative stain EM reconstruction of Dicer-PACT in the canonical conformation | |||||||||
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![]() | RNA-mediated gene silencing / pre-miRNA processing / RNaseIII | |||||||||
Function / homology | ![]() regulation of regulatory ncRNA processing / peripheral nervous system myelin formation / pre-miRNA binding / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / tRNA decay / global gene silencing by mRNA cleavage / negative regulation of Schwann cell proliferation / ribonuclease III / positive regulation of myelination ...regulation of regulatory ncRNA processing / peripheral nervous system myelin formation / pre-miRNA binding / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / tRNA decay / global gene silencing by mRNA cleavage / negative regulation of Schwann cell proliferation / ribonuclease III / positive regulation of myelination / apoptotic DNA fragmentation / nerve development / positive regulation of Schwann cell differentiation / RISC-loading complex / miRNA metabolic process / deoxyribonuclease I activity / RISC complex assembly / ribonuclease III activity / miRNA processing / siRNA binding / pre-miRNA processing / M-decay: degradation of maternal mRNAs by maternally stored factors / siRNA processing / outer ear morphogenesis / RISC complex / middle ear morphogenesis / skeletal system morphogenesis / MicroRNA (miRNA) biogenesis / protein kinase activator activity / antiviral innate immune response / negative regulation of tumor necrosis factor production / negative regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of intrinsic apoptotic signaling pathway / enzyme activator activity / RNA endonuclease activity / helicase activity / neuron projection morphogenesis / response to virus / PKR-mediated signaling / double-stranded RNA binding / cellular response to oxidative stress / protein stabilization / immune response / protein domain specific binding / negative regulation of cell population proliferation / negative regulation of gene expression / perinuclear region of cytoplasm / enzyme binding / negative regulation of transcription by RNA polymerase II / protein homodimerization activity / DNA binding / RNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 26.0 Å | |||||||||
![]() | Taylor DW / Ma E / Shigematsu H / Cianfrocco MA / Noland CL / Nagayama K / Nogales E / Doudna JA / Wang HW | |||||||||
![]() | ![]() Title: Substrate-specific structural rearrangements of human Dicer. Authors: David W Taylor / Enbo Ma / Hideki Shigematsu / Michael A Cianfrocco / Cameron L Noland / Kuniaki Nagayama / Eva Nogales / Jennifer A Doudna / Hong-Wei Wang / ![]() Abstract: Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor ...Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used EM and single-particle analysis of Dicer-RNA complexes to gain insight into the structural basis for human Dicer's substrate preference. Our studies show that Dicer traps pre-siRNAs in a nonproductive conformation, whereas interactions of Dicer with pre-miRNAs and dsRNA-binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 1.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.7 KB 11.7 KB | Display Display | ![]() |
Images | ![]() | 47.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 78.2 KB | Display | ![]() |
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Full document | ![]() | 77.3 KB | Display | |
Data in XML | ![]() | 494 B | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Negative stain EM reconstruction of Dicer-PACT in the canonical conformation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.36 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Human Dicer-PACT heterodimer in canonical conformation
Entire | Name: Human Dicer-PACT heterodimer in canonical conformation |
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Components |
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-Supramolecule #1000: Human Dicer-PACT heterodimer in canonical conformation
Supramolecule | Name: Human Dicer-PACT heterodimer in canonical conformation type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2 |
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Molecular weight | Theoretical: 260 KDa |
-Macromolecule #1: Endoribonuclease Dicer
Macromolecule | Name: Endoribonuclease Dicer / type: protein_or_peptide / ID: 1 / Name.synonym: Dicer, Helicase with RNase motif / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes / Database: NCBI |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 220 KDa |
Sequence | UniProtKB: Endoribonuclease Dicer / GO: pre-miRNA processing InterPro: Ribonuclease III domain, PAZ domain, Helicase, C-terminal, Helicase superfamily 1/2, ATP-binding domain, Double-stranded RNA-binding domain, INTERPRO: IPR001159, DEAD/DEAH box helicase ...InterPro: Ribonuclease III domain, PAZ domain, Helicase, C-terminal, Helicase superfamily 1/2, ATP-binding domain, Double-stranded RNA-binding domain, INTERPRO: IPR001159, DEAD/DEAH box helicase domain, Dicer dimerisation domain |
-Macromolecule #2: Interferon-inducible double stranded RNA-dependent protein kinase...
Macromolecule | Name: Interferon-inducible double stranded RNA-dependent protein kinase activator A type: protein_or_peptide / ID: 2 / Name.synonym: PACT / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes / Database: NCBI |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 34 KDa |
Sequence | UniProtKB: Interferon-inducible double-stranded RNA-dependent protein kinase activator A GO: double-stranded RNA binding InterPro: INTERPRO: IPR001159, Double-stranded RNA-binding domain |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 7.4 Details: 20 mM HEPES, pH 7.5, 150 mM KCl, 3 mM EDTA, 1 mM DTT, and 2.5% glycerol |
Staining | Type: NEGATIVE Details: After adsorption for 1 min, we stained the samples consecutively with three droplets of 2% (w/v) uranyl formate solution, blotted off the residual stain and air-dried the sample in a hood. |
Grid | Details: glow-discharged holey carbon grids with a thin layer of carbon over the holes |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | FEI TECNAI 12 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification |
Date | Jul 10, 2012 |
Image recording | Category: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 400 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 5.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: OTHER |
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Image processing
CTF correction | Details: each micrograph |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2/SPARX, multi-model / Number images used: 10000 |