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- EMDB-3066: A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain r... -

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Basic information

Entry
Database: EMDB / ID: EMD-3066
TitleA network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation
Map datacontour level in chimera
Sample
  • Sample: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1
  • Protein or peptide: SMG-1
  • Protein or peptide: SMG-8
  • Protein or peptide: SMG-9
  • Protein or peptide: UPF1
KeywordsCryo-electron microscopy / SMG-1 kinase / Phosphoinositide-3-kinase-like kinase / UPF1 substrate recruitment / SMG-8-9 kinase regulation / Nonsense-mediated mRNA decay
Function / homology
Function and homology information


double-stranded DNA helicase activity / supraspliceosomal complex / positive regulation of mRNA cis splicing, via spliceosome / exon-exon junction complex / telomere maintenance via semi-conservative replication / positive regulation of mRNA catabolic process / cell cycle phase transition / diacylglycerol-dependent serine/threonine kinase activity / regulation of translational termination / chromatoid body ...double-stranded DNA helicase activity / supraspliceosomal complex / positive regulation of mRNA cis splicing, via spliceosome / exon-exon junction complex / telomere maintenance via semi-conservative replication / positive regulation of mRNA catabolic process / cell cycle phase transition / diacylglycerol-dependent serine/threonine kinase activity / regulation of translational termination / chromatoid body / histone mRNA catabolic process / eye development / 3'-UTR-mediated mRNA destabilization / nuclear-transcribed mRNA catabolic process / regulation of telomere maintenance / regulation of protein kinase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / DNA duplex unwinding / telomeric DNA binding / phosphatidylinositol phosphate biosynthetic process / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / cellular response to interleukin-1 / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / mRNA export from nucleus / helicase activity / P-body / brain development / heart development / peptidyl-serine phosphorylation / DNA helicase / in utero embryonic development / cellular response to lipopolysaccharide / RNA helicase activity / DNA replication / chromosome, telomeric region / protein autophosphorylation / RNA helicase / non-specific serine/threonine protein kinase / protein kinase activity / DNA repair / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / chromatin binding / chromatin / protein-containing complex binding / negative regulation of apoptotic process / perinuclear region of cytoplasm / ATP hydrolysis activity / RNA binding / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Upf1 cysteine-histidine-rich (CH-rich) domain profile. / RNA helicase UPF1, 1B domain / RNA helicase (UPF2 interacting domain) / RNA helicase UPF1, 1B domain / RNA helicase UPF1, Cys/His rich zinc-binding domain / 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 ...Upf1 cysteine-histidine-rich (CH-rich) domain profile. / RNA helicase UPF1, 1B domain / RNA helicase (UPF2 interacting domain) / RNA helicase UPF1, 1B domain / RNA helicase UPF1, Cys/His rich zinc-binding domain / 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 / DNA2/NAM7-like helicase / : / DNA2/NAM7 helicase, helicase domain / AAA domain / Rapamycin binding domain / FATC domain / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / 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 / Armadillo-like helical / DNA2/NAM7 helicase-like, C-terminal / AAA domain / Armadillo-type fold / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nonsense-mediated mRNA decay factor SMG8 / Regulator of nonsense transcripts 1 / Serine/threonine-protein kinase SMG1 / Nonsense-mediated mRNA decay factor SMG9
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 19.0 Å
AuthorsDeniaud A / Karuppasamy M / Bock T / Masiulis S / Huard K / Garzoni F / Kerschgens K / Hentze MW / Kulozik AE / Beck M ...Deniaud A / Karuppasamy M / Bock T / Masiulis S / Huard K / Garzoni F / Kerschgens K / Hentze MW / Kulozik AE / Beck M / Neu-Yilik G / Schaffitzel C
CitationJournal: Nucleic Acids Res / Year: 2015
Title: A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation.
Authors: Aurélien Deniaud / Manikandan Karuppasamy / Thomas Bock / Simonas Masiulis / Karine Huard / Frédéric Garzoni / Kathrin Kerschgens / Matthias W Hentze / Andreas E Kulozik / Martin Beck / ...Authors: Aurélien Deniaud / Manikandan Karuppasamy / Thomas Bock / Simonas Masiulis / Karine Huard / Frédéric Garzoni / Kathrin Kerschgens / Matthias W Hentze / Andreas E Kulozik / Martin Beck / Gabriele Neu-Yilik / Christiane Schaffitzel /
Abstract: Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD ...Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1-8-9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1-8-9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1-8-9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.
History
DepositionJun 26, 2015-
Header (metadata) releaseJul 8, 2015-
Map releaseJul 15, 2015-
UpdateSep 9, 2015-
Current statusSep 9, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.047
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.047
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image_EMD_13...

Downloads & links

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Map

FileDownload / File: emd_3066.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcontour level in chimera
Voxel sizeX=Y=Z: 1.86 Å
Density
Contour LevelBy AUTHOR: 0.047 / Movie #1: 0.047
Minimum - Maximum-0.12514399 - 0.66809916
Average (Standard dev.)0.0002945 (±0.03465053)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 297.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.861.861.86
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z297.600297.600297.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1250.6680.000

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Supplemental data

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Sample components

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Entire : Complex of four proteins: SMG1, SMG8, SMG9 and UPF1

EntireName: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1
Components
  • Sample: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1
  • Protein or peptide: SMG-1
  • Protein or peptide: SMG-8
  • Protein or peptide: SMG-9
  • Protein or peptide: UPF1

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Supramolecule #1000: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1

SupramoleculeName: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1 / type: sample / ID: 1000
Details: The sample was monodisperse, and elution volume in size exclusion chromatography is consistent with an heterotetramer.
Oligomeric state: heterotetramer / Number unique components: 4
Molecular weightExperimental: 720 KDa / Theoretical: 720 KDa / Method: Size exclusion chromatography

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Macromolecule #1: SMG-1

MacromoleculeName: SMG-1 / type: protein_or_peptide / ID: 1 / Name.synonym: Serine/threonine-protein kinase SMG1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 420 KDa / Theoretical: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pLexm
SequenceUniProtKB: Serine/threonine-protein kinase SMG1

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Macromolecule #2: SMG-8

MacromoleculeName: SMG-8 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 110 KDa / Theoretical: 110 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pLexm
SequenceUniProtKB: Nonsense-mediated mRNA decay factor SMG8

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Macromolecule #3: SMG-9

MacromoleculeName: SMG-9 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 60 KDa / Theoretical: 60 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pLexm
SequenceUniProtKB: Nonsense-mediated mRNA decay factor SMG9

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Macromolecule #4: UPF1

MacromoleculeName: UPF1 / type: protein_or_peptide / ID: 4 / Name.synonym: RENT1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 130 KDa / Theoretical: 130 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf21 / Recombinant plasmid: pFastBBac
SequenceUniProtKB: Regulator of nonsense transcripts 1

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 9
Details: 20 mM Tris pH 9.0, 100 mM KCl, 25 mM glycine, 1 mM DTT and 2 mM biotin
StainingType: NEGATIVE / Details: cryo
GridDetails: copper 300 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: 2 second blotting and blot force of 3

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.3 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Specialist opticsEnergy filter - Name: not used
Sample stageSpecimen holder: FEI polara multispecimen holder, nitrogen cooled
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 86 K
DateJan 30, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 1300 / Average electron dose: 13 e/Å2 / Bits/pixel: 32
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: xmipp / Number images used: 10451

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