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- EMDB-2665: Structure of SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SM... -

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Basic information

Entry
Database: EMDB / ID: EMD-2665
TitleStructure of SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SMG9 and UPF2
Map dataRecosntruction of the SMG1C-UPF2 complex, comprising SMG1, SMG8, SMG9 and UPF2
Sample
  • Sample: SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SMG9 and UPF2
  • Protein or peptide: Serine/threonine-protein kinase SMG1
  • Protein or peptide: SMG8
  • Protein or peptide: SMG9
  • Protein or peptide: UPF2
KeywordsNMD / SMG1 / SMG8 / SMG9 / UPF2 / PIKK / RNA degradation
Function / homology
Function and homology information


RNA metabolic process / exon-exon junction complex / 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 ...RNA metabolic process / exon-exon junction complex / 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 / animal organ regeneration / mRNA export from nucleus / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / liver development / brain development / Regulation of expression of SLITs and ROBOs / cytoplasmic ribonucleoprotein granule / heart development / peptidyl-serine phosphorylation / in utero embryonic development / protein autophosphorylation / non-specific serine/threonine protein kinase / protein kinase activity / protein serine kinase activity / DNA repair / protein serine/threonine kinase activity / DNA damage response / negative regulation of apoptotic process / perinuclear region of cytoplasm / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
: / Nonsense-mediated mRNA decay factor SMG8 / Up-frameshift suppressor 2, C-terminal / Up-frameshift suppressor 2, C-terminal / Nonsense-mediated mRNA decay protein Nmd2/UPF2 / Up-frameshift suppressor 2 / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Nonsense-mediated mRNA decay factor SMG8/SMG9 / Smg8_Smg9 / Nonsense-mediated mRNA decay factor SMG9 ...: / Nonsense-mediated mRNA decay factor SMG8 / Up-frameshift suppressor 2, C-terminal / Up-frameshift suppressor 2, C-terminal / Nonsense-mediated mRNA decay protein Nmd2/UPF2 / Up-frameshift suppressor 2 / 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 / MIF4G domain / Rapamycin binding domain / Middle domain of eukaryotic initiation factor 4G (eIF4G) / MIF4G-like, type 3 / MIF4G-like, type 3 / : / FATC domain / FATC / FAT domain profile. / FATC domain profile. / FATC domain / PIK-related kinase / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Armadillo-like helical / Armadillo-type fold / Armadillo-type fold / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nonsense-mediated mRNA decay factor SMG8 / Serine/threonine-protein kinase SMG1 / Nonsense-mediated mRNA decay factor SMG9 / Regulator of nonsense transcripts 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 21.7 Å
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.
History
DepositionMay 27, 2014-
Header (metadata) releaseJun 18, 2014-
Map releaseJul 9, 2014-
UpdateAug 20, 2014-
Current statusAug 20, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.76
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.76
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2665.png

Downloads & links

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Map

FileDownload / File: emd_2665.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRecosntruction of the SMG1C-UPF2 complex, comprising SMG1, SMG8, SMG9 and UPF2
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.76 / Movie #1: 4.76
Minimum - Maximum-6.37496376 - 14.724467280000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-72-72-72
Dimensions144144144
Spacing144144144
CellA=B=C: 408.96 Å
α=β=γ: 90.0 °

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/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-6.37514.7240.000

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Supplemental data

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Sample components

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Entire : SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SMG9 and UPF2

EntireName: SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SMG9 and UPF2
Components
  • Sample: SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SMG9 and UPF2
  • Protein or peptide: Serine/threonine-protein kinase SMG1
  • Protein or peptide: SMG8
  • Protein or peptide: SMG9
  • Protein or peptide: UPF2

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Supramolecule #1000: SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SMG9 and UPF2

SupramoleculeName: SMG1C-UPF2 complex, comprising SMG1 kinase, SMG8, SMG9 and UPF2
type: sample / ID: 1000 / Number unique components: 4
Molecular weightTheoretical: 730 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: UPF2

MacromoleculeName: UPF2 / type: protein_or_peptide / ID: 4 / Name.synonym: RENT2_HUMAN / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 148 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Regulator of nonsense transcripts 2
GO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
InterPro: Armadillo-type fold, INTERPRO: IPR016021, MIF4G-like, type 3, Up-frameshift suppressor 2, C-terminal

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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% 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)
DateMay 10, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Number real images: 465 / 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: 21.7 Å / Resolution method: OTHER / Software - Name: EMAN, EMAN2, Xmipp / Number images used: 18716
Final two d classificationNumber classes: 490

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Atomic model buiding 1

Initial modelPDB ID:

4jsp

SoftwareName: Chimera
DetailsThe structure was separately fitted using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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Atomic model buiding 2

Initial modelPDB ID:

3kgv

SoftwareName: Chimera
DetailsHEAT repeat regions from DNA-PKcs were separately fitted into SMG1 using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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