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- EMDB-3279: Electron microscopy of human RNA helicase DHX34 -

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Basic information

Entry
Database: EMDB / ID: EMD-3279
TitleElectron microscopy of human RNA helicase DHX34
Map dataReconstruction of DHX34
Sample
  • Sample: DHX34
  • Protein or peptide: DHX34
KeywordsNMD / SMG1 / SMG8 / SMG9 / PIKK / DHX34 / RNA degradation / RNA helicase
Function / homology
Function and homology information


RNA helicase activity => GO:0003724 / RNA binding => GO:0003723 / negative regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / nuclear-transcribed mRNA catabolic process / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / RNA processing / positive regulation of phosphorylation / RNA helicase activity / RNA helicase / protein-containing complex binding ...RNA helicase activity => GO:0003724 / RNA binding => GO:0003723 / negative regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / nuclear-transcribed mRNA catabolic process / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / RNA processing / positive regulation of phosphorylation / RNA helicase activity / RNA helicase / protein-containing complex binding / ATP hydrolysis activity / RNA binding / ATP binding / membrane / cytoplasm
Similarity search - Function
: / DEAD-box helicase, OB fold / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase-associated domain / Helicase-associated domain / Helicase associated domain (HA2), winged-helix / Helicase associated domain (HA2) Add an annotation / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain ...: / DEAD-box helicase, OB fold / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase-associated domain / Helicase-associated domain / Helicase associated domain (HA2), winged-helix / Helicase associated domain (HA2) Add an annotation / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Probable ATP-dependent RNA helicase DHX34
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 24.23 Å
AuthorsMelero R / Hug N / Lopez-Perrote A / Yamashita A / Caceres J / Llorca O
CitationJournal: Nat Commun / Year: 2016
Title: The RNA helicase DHX34 functions as a scaffold for SMG1-mediated UPF1 phosphorylation.
Authors: Roberto Melero / Nele Hug / Andrés López-Perrote / Akio Yamashita / Javier F Cáceres / Oscar Llorca /
Abstract: Nonsense-mediated decay (NMD) is a messenger RNA quality-control pathway triggered by SMG1-mediated phosphorylation of the NMD factor UPF1. In recent times, the RNA helicase DHX34 was found to ...Nonsense-mediated decay (NMD) is a messenger RNA quality-control pathway triggered by SMG1-mediated phosphorylation of the NMD factor UPF1. In recent times, the RNA helicase DHX34 was found to promote mRNP remodelling, leading to activation of NMD. Here we demonstrate the mechanism by which DHX34 functions in concert with SMG1. DHX34 comprises two distinct structural units, a core that binds UPF1 and a protruding carboxy-terminal domain (CTD) that binds the SMG1 kinase, as shown using truncated forms of DHX34 and electron microscopy of the SMG1-DHX34 complex. Truncation of the DHX34 CTD does not affect binding to UPF1; however, it compromises DHX34 binding to SMG1 to affect UPF1 phosphorylation and hence abrogate NMD. Altogether, these data suggest the existence of a complex comprising SMG1, UPF1 and DHX34, with DHX34 functioning as a scaffold for UPF1 and SMG1. This complex promotes UPF1 phosphorylation leading to functional NMD.
History
DepositionDec 11, 2015-
Header (metadata) releaseJan 13, 2016-
Map releaseJan 13, 2016-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3279_DHX...

Downloads & links

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Map

FileDownload / File: emd_3279.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of DHX34
Voxel sizeX=Y=Z: 5.68 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.04734212 - 0.21498932
Average (Standard dev.)-0.00044793 (±0.01133275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 408.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.685.685.68
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z408.960408.960408.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-0.0470.215-0.000

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Supplemental data

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Sample components

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Entire : DHX34

EntireName: DHX34
Components
  • Sample: DHX34
  • Protein or peptide: DHX34

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Supramolecule #1000: DHX34

SupramoleculeName: DHX34 / type: sample / ID: 1000 / Oligomeric state: Monomeric DHX34 / Number unique components: 1
Molecular weightTheoretical: 128 KDa

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Macromolecule #1: DHX34

MacromoleculeName: DHX34 / type: protein_or_peptide / ID: 1 / Name.synonym: DHX34 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 128 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293T cells
SequenceUniProtKB: Probable ATP-dependent RNA helicase DHX34
GO: cytoplasm, membrane, ATP binding, RNA helicase activity => GO:0003724, RNA binding => GO:0003723, negative regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, ...GO: cytoplasm, membrane, ATP binding, RNA helicase activity => GO:0003724, RNA binding => GO:0003723, negative regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, nuclear-transcribed mRNA catabolic process, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, RNA processing
InterPro: DEAD-box helicase, OB fold, Helicase-associated domain, Helicase superfamily 1/2, ATP-binding domain, Helicase, C-terminal, P-loop containing nucleoside triphosphate hydrolase, INTERPRO: IPR015880

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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: 7.5
Details: 10 mM HEPES-KOH, 150 mM NaCl, 20% (v/v) glycerol, 10 mM MgCl2
StainingType: NEGATIVE / Details: 1% uranyl formate
GridDetails: 400 mesh grid with thin carbon support, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1230
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 54926 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.9 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: JEOL
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using a TVIPS F416 CMOS and the EM-MENU software (TVIPS)
DateFeb 17, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Average electron dose: 15 e/Å2
Details: Using a TVIPS F416 CMOS and the EM-TOOLS software (TVIPS)
Bits/pixel: 16

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Image processing

CTF correctionDetails: Each micrograph using BSOFT
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.23 Å / Resolution method: OTHER / Software - Name: EMAN, EMAN2, Xmipp / Number images used: 12316

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