+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5603 | |||||||||
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Title | Substrate-specific structural rearrangements of human Dicer | |||||||||
Map data | Zernike phase-contrast cryo-EM reconstruction of Dicer-pre-let7 | |||||||||
Sample |
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Keywords | RNA-mediated gene silencing / pre-miRNA processing / RNaseIII | |||||||||
Function / homology | Function and homology information peripheral nervous system myelin formation / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / global gene silencing by mRNA cleavage / tRNA decay / pre-miRNA binding / Small interfering RNA (siRNA) biogenesis / negative regulation of Schwann cell proliferation / positive regulation of myelination / ribonuclease III / deoxyribonuclease I activity ...peripheral nervous system myelin formation / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / global gene silencing by mRNA cleavage / tRNA decay / pre-miRNA binding / Small interfering RNA (siRNA) biogenesis / negative regulation of Schwann cell proliferation / positive regulation of myelination / ribonuclease III / deoxyribonuclease I activity / apoptotic DNA fragmentation / miRNA metabolic process / positive regulation of Schwann cell differentiation / nerve development / RISC-loading complex / RISC complex assembly / miRNA processing / pre-miRNA processing / ribonuclease III activity / siRNA processing / siRNA binding / M-decay: degradation of maternal mRNAs by maternally stored factors / RISC complex / MicroRNA (miRNA) biogenesis / negative regulation of tumor necrosis factor production / negative regulation of tumor necrosis factor-mediated signaling pathway / RNA endonuclease activity / neuron projection morphogenesis / helicase activity / double-stranded RNA binding / protein domain specific binding / negative regulation of gene expression / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / DNA binding / RNA binding / extracellular exosome / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 31.0 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_5603.map.gz | 2.5 MB | EMDB map data format | |
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Header (meta data) | emd-5603-v30.xml emd-5603.xml | 12 KB 12 KB | Display Display | EMDB header |
Images | emd_5603.png | 120.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5603 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5603 | 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_5603.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Zernike phase-contrast cryo-EM reconstruction of Dicer-pre-let7 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.07 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 in complex with pre-let7
Entire | Name: Human Dicer in complex with pre-let7 |
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Components |
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-Supramolecule #1000: Human Dicer in complex with pre-let7
Supramolecule | Name: Human Dicer in complex with pre-let7 / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2 |
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Molecular weight | Theoretical: 250 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: pre-let7
Macromolecule | Name: pre-let7 / type: other / ID: 2 / Classification: OTHER_NA / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Theoretical: 24 KDa |
Sequence | String: UGAGGUAGUA GGUUGUAUAG UUUUAGGGUC ACACCCACCA CUGGGAGAUA ACUAUACAAU CUACUGUCUU ACC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.05 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 |
Grid | Details: glow-discharged Quantifoil R 1.2/1.3 MO 200 mesh holey carbon grids |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV Method: The samples were automatically blotted for 4-5 s at -2.5 mm offset before plunging. |
-Electron microscopy
Microscope | JEOL 3100FFC |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.7 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Nominal magnification: 60000 |
Specialist optics | Energy filter - Name: JEOL / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Sample stage | Specimen holder: Liquid helium cooled stage maintained at 55 K Specimen holder model: JEOL |
Temperature | Min: 45 K / Max: 60 K / Average: 55 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification |
Date | Jan 10, 2010 |
Image recording | Category: CCD / Film or detector model: GENERIC GATAN (2k x 2k) / Number real images: 800 / Average electron dose: 20 e/Å2 |
-Image processing
CTF correction | Details: each micrograph |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 31.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2/SPARX / Number images used: 4100 |