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- EMDB-2663: Structure of SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9 -

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Entry
Database: EMDB / ID: 2663
TitleStructure of SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9
Map dataRecosntruction of the SMG1C complex, comprising SMG1, SMG8 and SMG9
SampleSMG1C complex, comprising SMG1 kinase, SMG8 and SMG9:
Serine/threonine-protein kinase SMG1 / SMG8 / SMG9
KeywordsNMD / SMG1 / SMG8 / SMG9 / PIKK / RNA degradation
Function / homologyPhosphatidylinositol 3- and 4-kinase / Serine/threonine-protein kinase SMG1 / FATC domain / Smg8_Smg9 / Protein kinase-like domain superfamily / PIK-related kinase / FATC domain / Serine/threonine-protein kinase smg-1 / Armadillo-type fold / Phosphatidylinositol 3/4-kinase, conserved site ...Phosphatidylinositol 3- and 4-kinase / Serine/threonine-protein kinase SMG1 / FATC domain / Smg8_Smg9 / Protein kinase-like domain superfamily / PIK-related kinase / FATC domain / Serine/threonine-protein kinase smg-1 / Armadillo-type fold / Phosphatidylinositol 3/4-kinase, conserved site / Smg8/Smg9 / Phosphatidylinositol 3-/4-kinase, catalytic domain / Protein SMG8 / Serine/threonine-protein kinase SMG1, N-terminal / Serine/threonine-protein kinase SMG1 N-terminal / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3- and 4-kinases family profile. / FAT domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / FATC domain profile. / Protein SMG9 / SMG1, PIKK catalytic domain / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Smg8/Smg9 / Protein SMG8 / eye development / regulation of telomere maintenance / RNA metabolic process / regulation of response to DNA damage stimulus / regulation of protein kinase activity / telomeric DNA binding / phosphatidylinositol phosphorylation / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / mRNA export from nucleus / brain development / intracellular / heart development / peptidyl-serine phosphorylation / non-specific serine/threonine protein kinase / protein kinase activity / protein autophosphorylation / DNA repair / protein serine/threonine kinase activity / P-loop containing nucleoside triphosphate hydrolase / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm / Protein SMG8 / Serine/threonine-protein kinase SMG1 / Protein SMG9
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / 20.8 Å resolution
AuthorsMelero R / Uchiyama A / Castano R / Kataoka N / Kurosawa H / Ohno S / Yamashita A / Llorca O
CitationJournal: Structure / Year: 2014
Title: Structures of SMG1-UPFs complexes: SMG1 contributes to regulate UPF2-dependent activation of UPF1 in NMD.
Authors: Roberto Melero / Akiko Uchiyama / Raquel Castaño / Naoyuki Kataoka / Hitomi Kurosawa / Shigeo Ohno / Akio Yamashita / Oscar Llorca
Abstract: SMG1, a PI3K-related kinase, plays a critical role in nonsense-mediated mRNA decay (NMD) in mammals. SMG1-mediated phosphorylation of the UPF1 helicase is an essential step during NMD initiation. ...SMG1, a PI3K-related kinase, plays a critical role in nonsense-mediated mRNA decay (NMD) in mammals. SMG1-mediated phosphorylation of the UPF1 helicase is an essential step during NMD initiation. Both SMG1 and UPF1 are presumably activated by UPF2, but this regulation is incompletely understood. Here we reveal that SMG1C (a complex containing SMG1, SMG8, and SMG9) contributes to regulate NMD by recruiting UPF1 and UPF2 to distinct sites in the vicinity of the kinase domain. UPF2 binds SMG1 in an UPF1-independent manner in vivo, and the SMG1C-UPF2 structure shows UPF2 recognizes the FRB domain, a region that regulates the related mTOR kinase. The molecular architectures of several SMG1C-UPFs complexes, obtained by combining electron microscopy with in vivo and in vitro interaction analyses, competition experiments, and mutations, suggest that UPF2 can be transferred to UPF1 within SMG1C, inducing UPF2-dependent conformational changes required to activate UPF1 within an SMG1C-UPF1-UPF2 complex.
DateDeposition: May 27, 2014 / Header (metadata) release: Jun 18, 2014 / Map release: Jul 9, 2014 / Last update: Aug 20, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2663.map.gz (map file in CCP4 format, 11665 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
144 pix
2.84 Å/pix.
= 408.96 Å
144 pix
2.84 Å/pix.
= 408.96 Å
144 pix
2.84 Å/pix.
= 408.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.84 Å
Density
Contour Level:3.4 (by author), 3.4 (movie #1):
Minimum - Maximum-2.93224406 - 11.79192543
Average (Standard dev.)-0.00040864 (0.72179729)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions144144144
Origin000
Limit143143143
Spacing144144144
CellA=B=C: 408.96 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.842.842.84
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z408.960408.960408.960
α/β/γ90.00090.00090.000
start NX/NY/NZ0-51-100
NX/NY/NZ82103201
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-2.93211.792-0.000

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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

EntireName: SMG1C complex, comprising SMG1 kinase, SMG8 and SMG9 / Number of components: 3
MassTheoretical: 580 kDa

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Component #1: protein, Serine/threonine-protein kinase SMG1

ProteinName: Serine/threonine-protein kinase SMG1 / a.k.a: SMG-1 / Recombinant expression: Yes
MassTheoretical: 410 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: 293T cells
External referencesUniProt: Serine/threonine-protein kinase SMG1
Gene Ontology: DNA repair, RNA metabolic process, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, ATP binding

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Component #2: protein, SMG8

ProteinName: SMG8 / a.k.a: SMG-8 / Recombinant expression: Yes
MassTheoretical: 109 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: 293T cells
External referencesInterPro: Protein SMG8
Gene Ontology: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
UniProt: Protein SMG8

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Component #3: protein, SMG9

ProteinName: SMG9 / a.k.a: SMG-9 / Recombinant expression: Yes
MassTheoretical: 60 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: 293T cells
External referencesUniProt: Protein SMG9
InterPro: P-loop containing nucleoside triphosphate hydrolase, Smg8/Smg9
Gene Ontology: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.01 mg/ml
Buffer solution: 10 mM HEPES-KOH, 150 mM NaCl, 20% glycerol, 10 mM MgCl2
pH: 7.5
Support film400 mesh grid with thin carbon support, glow discharged
Staining1% uranyl formate
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 1230 / Date: Apr 5, 2012
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 100 kV / Electron dose: 15 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 40000 X (nominal), 54926 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected using a TVIPS F416 CMOS and the EM-MENU software (TVIPS)
Cs: 2.9 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2500 nm
Specimen HolderModel: JEOL
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 542 / Sampling size: 15.6 microns / Bit depth: 16
Details: Using a TVIPS F416 CMOS and the EM-TOOLS software (TVIPS)

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 490 / Applied symmetry: C1 (asymmetric) / Number of projections: 21020
3D reconstructionAlgorithm: Angular Refinement / Software: EMAN, EMAN2, Xmipp / CTF correction: Each micrograph using BSOFT / Resolution: 20.8 Å / Resolution method: FSC 0.5, semi-independent

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Target criteria: Correlation coefficient / Refinement space: REAL / Details: The structure was separately fitted using Chimera
Input PDB model: 4JSP
Modeling #2Software: Chimera / Refinement protocol: rigid body / Target criteria: Correlation coefficient / Refinement space: REAL
Details: HEAT repeat regions from DNA-PKcs were separately fitted into SMG1 using Chimera
Input PDB model: 3KGV

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