+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5606 | |||||||||
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Title | Substrate-specific structural rearrangements of human Dicer | |||||||||
Map data | Negative stain EM reconstruction of Dicer-PACT in the open conformation | |||||||||
Sample |
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Keywords | RNA-mediated gene silencing / pre-miRNA processing / RNaseIII | |||||||||
Function / homology | Function and homology information regulation of regulatory ncRNA processing / peripheral nervous system myelin formation / pre-miRNA binding / global gene silencing by mRNA cleavage / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / tRNA decay / negative regulation of Schwann cell proliferation / positive regulation of myelination / apoptotic DNA fragmentation ...regulation of regulatory ncRNA processing / peripheral nervous system myelin formation / pre-miRNA binding / global gene silencing by mRNA cleavage / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / tRNA decay / negative regulation of Schwann cell proliferation / positive regulation of myelination / apoptotic DNA fragmentation / ribonuclease III / deoxyribonuclease I activity / nerve development / positive regulation of Schwann cell differentiation / RISC-loading complex / miRNA metabolic process / RISC complex assembly / miRNA processing / ribonuclease III activity / pre-miRNA processing / siRNA binding / siRNA processing / Regulation of MITF-M-dependent genes involved in apoptosis / M-decay: degradation of maternal mRNAs by maternally stored factors / RISC complex / outer ear morphogenesis / middle ear morphogenesis / skeletal system morphogenesis / MicroRNA (miRNA) biogenesis / protein kinase activator activity / negative regulation of tumor necrosis factor production / negative regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of intrinsic apoptotic signaling pathway / antiviral innate immune response / enzyme activator activity / RNA endonuclease activity / neuron projection morphogenesis / helicase activity / 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 / RNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 26.0 Å | |||||||||
Authors | Taylor DW / Ma E / Shigematsu H / Cianfrocco MA / Noland CL / Nagayama K / Nogales E / Doudna JA / Wang HW | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2013 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 |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5606.map.gz | 1.7 MB | EMDB map data format | |
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Header (meta data) | emd-5606-v30.xml emd-5606.xml | 11.7 KB 11.7 KB | Display Display | EMDB header |
Images | emd_5606.png | 54.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5606 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5606 | HTTPS FTP |
-Validation report
Summary document | emd_5606_validation.pdf.gz | 78.2 KB | Display | EMDB validaton report |
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Full document | emd_5606_full_validation.pdf.gz | 77.3 KB | Display | |
Data in XML | emd_5606_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5606 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5606 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_5606.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Negative stain EM reconstruction of Dicer-PACT in the open conformation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
-Sample components
-Entire : Human Dicer-PACT heterodimer in open conformation
Entire | Name: Human Dicer-PACT heterodimer in open conformation |
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Components |
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-Supramolecule #1000: Human Dicer-PACT heterodimer in open conformation
Supramolecule | Name: Human Dicer-PACT heterodimer in open 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: Homo sapiens (human) / synonym: Human |
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: Homo sapiens (human) / synonym: Human |
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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Processing | single particle reconstruction |
Aggregation state | particle |
-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 |
-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 |
-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 |