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- EMDB-5606: Substrate-specific structural rearrangements of human Dicer -

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Basic information

Entry
Database: EMDB / ID: EMD-5606
TitleSubstrate-specific structural rearrangements of human Dicer
Map dataNegative stain EM reconstruction of Dicer-PACT in the open conformation
Sample
  • Sample: Human Dicer-PACT heterodimer in open conformation
  • Protein or peptide: Endoribonuclease DicerDicer
  • Protein or peptide: Interferon-inducible double stranded RNA-dependent protein kinase activator A
KeywordsRNA-mediated gene silencing / pre-miRNA processing / RNaseIII
Function / homology
Function and homology information


peripheral nervous system myelin formation / regulation of regulatory ncRNA processing / 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 ...peripheral nervous system myelin formation / regulation of regulatory ncRNA processing / 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 / outer ear morphogenesis / middle ear morphogenesis / skeletal system morphogenesis / MicroRNA (miRNA) biogenesis / negative regulation of tumor necrosis factor production / enzyme activator activity / negative regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of intrinsic apoptotic signaling pathway / 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 / DNA binding / RNA binding / extracellular exosome / nucleoplasm / ATP binding / membrane / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
PRKRA, first double-stranded RNA binding domain / PRKRA, second double-stranded RNA binding domain / PRKRA, third double-stranded RNA binding domain / : / Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / Dicer, platform domain ...PRKRA, first double-stranded RNA binding domain / PRKRA, second double-stranded RNA binding domain / PRKRA, 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 / Dicer dimerisation domain / Dicer dimerisation domain superfamily / 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 / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain / PAZ domain / PAZ domain profile. / Double-stranded RNA binding motif / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / Double-stranded RNA-binding domain / DEAD/DEAH box helicase domain / 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, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Interferon-inducible double-stranded RNA-dependent protein kinase activator A / Endoribonuclease Dicer
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsTaylor DW / Ma E / Shigematsu H / Cianfrocco MA / Noland CL / Nagayama K / Nogales E / Doudna JA / Wang HW
CitationJournal: 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
DepositionMar 10, 2013-
Header (metadata) releaseMar 20, 2013-
Map releaseMay 1, 2013-
UpdateJun 19, 2013-
Current statusJun 19, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.83
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.83
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5606.png

Downloads & links

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Map

FileDownload / File: emd_5606.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM reconstruction of Dicer-PACT in the open conformation
Voxel sizeX=Y=Z: 4.36 Å
Density
Contour LevelBy AUTHOR: 4.83 / Movie #1: 4.83
Minimum - Maximum-4.73826694 - 19.24861336
Average (Standard dev.)0.0 (±0.99999905)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 348.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-4.73819.2490.000

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Supplemental data

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Sample components

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Entire : Human Dicer-PACT heterodimer in open conformation

EntireName: Human Dicer-PACT heterodimer in open conformation
Components
  • Sample: Human Dicer-PACT heterodimer in open conformation
  • Protein or peptide: Endoribonuclease DicerDicer
  • Protein or peptide: Interferon-inducible double stranded RNA-dependent protein kinase activator A

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Supramolecule #1000: Human Dicer-PACT heterodimer in open conformation

SupramoleculeName: Human Dicer-PACT heterodimer in open conformation / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: Endoribonuclease Dicer

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 220 KDa
SequenceUniProtKB: 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

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Macromolecule #2: Interferon-inducible double stranded RNA-dependent protein kinase...

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 34 KDa
SequenceUniProtKB: Interferon-inducible double-stranded RNA-dependent protein kinase activator A
GO: double-stranded RNA binding
InterPro: INTERPRO: IPR001159, Double-stranded RNA-binding domain

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.4
Details: 20 mM HEPES, pH 7.5, 150 mM KCl, 3 mM EDTA, 1 mM DTT, and 2.5% glycerol
StainingType: 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.
GridDetails: glow-discharged holey carbon grids with a thin layer of carbon over the holes
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 5.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateJul 10, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 400 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionAlgorithm: 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

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