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- EMDB-3278: Electron microscopy of the SMG1C-DHX34 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3278
TitleElectron microscopy of the SMG1C-DHX34 complex
Map dataReconstruction of SMG1C-DHX34
Sample
  • Sample: SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9, bound to the DHX34 helicase
  • Protein or peptide: Serine/threonine-protein kinase SMG1
  • Protein or peptide: SMG8
  • Protein or peptide: SMG9
  • 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 / RNA metabolic process / regulation of protein kinase activity / diacylglycerol-dependent serine/threonine kinase activity / chromatoid body / eye development / regulation of telomere maintenance / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay ...RNA helicase activity => GO:0003724 / RNA binding => GO:0003723 / negative regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / RNA metabolic process / regulation of protein kinase activity / diacylglycerol-dependent serine/threonine kinase activity / chromatoid body / eye development / regulation of telomere maintenance / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / telomeric DNA binding / phosphatidylinositol phosphate biosynthetic process / nuclear-transcribed mRNA catabolic process / mRNA export from nucleus / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / RNA processing / positive regulation of phosphorylation / helicase activity / brain development / heart development / peptidyl-serine phosphorylation / in utero embryonic development / protein autophosphorylation / RNA helicase activity / non-specific serine/threonine protein kinase / protein kinase activity / RNA helicase / protein serine kinase activity / DNA repair / protein serine/threonine kinase activity / DNA damage response / protein-containing complex binding / negative regulation of apoptotic process / ATP hydrolysis activity / RNA binding / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
: / Nonsense-mediated mRNA decay factor SMG8 / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Smg8_Smg9 / Nonsense-mediated mRNA decay factor SMG9 / Serine/threonine-protein kinase SMG1 / Serine/threonine-protein kinase SMG1, N-terminal / SMG1, PIKK catalytic domain / Serine/threonine-protein kinase smg-1 ...: / Nonsense-mediated mRNA decay factor SMG8 / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Smg8_Smg9 / Nonsense-mediated mRNA decay factor SMG9 / Serine/threonine-protein kinase SMG1 / Serine/threonine-protein kinase SMG1, N-terminal / SMG1, PIKK catalytic domain / Serine/threonine-protein kinase smg-1 / Serine/threonine-protein kinase SMG1 N-terminal / Rapamycin binding domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix / Helicase-associated domain / Helicase-associated domain / Helicase associated domain (HA2) Add an annotation / : / FATC domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / Armadillo-like helical / 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 / Armadillo-type fold / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Probable ATP-dependent RNA helicase DHX34 / Nonsense-mediated mRNA decay factor SMG8 / Serine/threonine-protein kinase SMG1 / Nonsense-mediated mRNA decay factor SMG9
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 20.89 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: 0.055
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3278_SMG...

Downloads & links

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Map

FileDownload / File: emd_3278.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of SMG1C-DHX34
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.68 Å/pix.
x 72 pix.
= 408.96 Å		5.68 Å/pix.
x 72 pix.
= 408.96 Å		5.68 Å/pix.
x 72 pix.
= 408.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.68 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.04281452 - 0.16968802
Average (Standard dev.)0.00027362 (±0.01273971)
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-0.0430.1700.000

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Supplemental data

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Sample components

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Entire : SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9, bound to th...

EntireName: SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9, bound to the DHX34 helicase
Components
  • Sample: SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9, bound to the DHX34 helicase
  • Protein or peptide: Serine/threonine-protein kinase SMG1
  • Protein or peptide: SMG8
  • Protein or peptide: SMG9
  • Protein or peptide: DHX34

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Supramolecule #1000: SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9, bound to th...

SupramoleculeName: SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9, bound to the DHX34 helicase
type: sample / ID: 1000
Oligomeric state: Heterotetramer of SMG1, SMG8, SMG9 and DHX34
Number unique components: 4
Molecular weightTheoretical: 770 KDa

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Macromolecule #1: Serine/threonine-protein kinase SMG1

MacromoleculeName: Serine/threonine-protein kinase SMG1 / type: protein_or_peptide / ID: 1 / Name.synonym: SMG-1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 410 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293T cells
SequenceUniProtKB: Serine/threonine-protein kinase SMG1
GO: DNA repair, RNA metabolic process, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, ATP binding

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Macromolecule #2: SMG8

MacromoleculeName: SMG8 / type: protein_or_peptide / ID: 2 / Name.synonym: SMG-8 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 109 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293T cells
SequenceUniProtKB: Nonsense-mediated mRNA decay factor SMG8
GO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: Nonsense-mediated mRNA decay factor SMG8

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Macromolecule #3: SMG9

MacromoleculeName: SMG9 / type: protein_or_peptide / ID: 3 / Name.synonym: SMG-9 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 60 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293T cells
SequenceUniProtKB: Nonsense-mediated mRNA decay factor SMG9
GO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: P-loop containing nucleoside triphosphate hydrolase, Nonsense-mediated mRNA decay factor SMG8/SMG9

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Macromolecule #4: DHX34

MacromoleculeName: DHX34 / type: protein_or_peptide / ID: 4 / 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
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using a TVIPS F416 CMOS and the EM-MENU software (TVIPS)
DateJul 25, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Number real images: 542 / Average electron dose: 15 e/Å2
Details: Using a TVIPS F416 CMOS and the EM-TOOLS software (TVIPS)
Bits/pixel: 16
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 54926 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.9 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: JEOL

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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: 20.89 Å / Resolution method: OTHER / Software - Name: EMAN, EMAN2, Xmipp / Number images used: 21020
Final two d classificationNumber classes: 490

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