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- EMDB-1852: EM map of the negative stained SMG-1-8-9 complex. -

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Basic information

Entry
Database: EMDB / ID: EMD-1852
TitleEM map of the negative stained SMG-1-8-9 complex.
Map dataThis is the EM map of the negatively stained SMG-1-8-9 complex
Sample
  • Sample: Human SMG-1 kinase bound to SMG-9 and SMG-8
  • Protein or peptide: SMG-1
  • Protein or peptide: SMG-9
  • Protein or peptide: SMG-8
KeywordsNonsense-mediated mRNA decay / NMD / SMG-1 / SMG-8 / SMG-9 / SMG1C
Function / homologynuclear-transcribed mRNA catabolic process, nonsense-mediated decay
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsArias-Palomo E / Yamashita A / Fernandez IS / Nunez-Ramirez R / Bamba Y / Izumi N / Ohno S / Llorca O
CitationJournal: Genes Dev / Year: 2011
Title: The nonsense-mediated mRNA decay SMG-1 kinase is regulated by large-scale conformational changes controlled by SMG-8.
Authors: Ernesto Arias-Palomo / Akio Yamashita / Israel S Fernández / Rafael Núñez-Ramírez / Yumi Bamba / Natsuko Izumi / Shigeo Ohno / Oscar Llorca /
Abstract: Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression ...Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD.
History
DepositionDec 17, 2010-
Header (metadata) releaseJan 21, 2011-
Map releaseDec 23, 2011-
UpdateDec 23, 2011-
Current statusDec 23, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1852.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the EM map of the negatively stained SMG-1-8-9 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.2 Å/pix.
x 80 pix.
= 336. Å
4.2 Å/pix.
x 80 pix.
= 336. Å
4.2 Å/pix.
x 80 pix.
= 336. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.2 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.0221306 - 0.179076
Average (Standard dev.)0.00185082 (±0.0110756)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 336 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z336.000336.000336.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-30-24-70
NX/NY/NZ6149141
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-0.0220.1790.002

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Supplemental data

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Sample components

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Entire : Human SMG-1 kinase bound to SMG-9 and SMG-8

EntireName: Human SMG-1 kinase bound to SMG-9 and SMG-8
Components
  • Sample: Human SMG-1 kinase bound to SMG-9 and SMG-8
  • Protein or peptide: SMG-1
  • Protein or peptide: SMG-9
  • Protein or peptide: SMG-8

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Supramolecule #1000: Human SMG-1 kinase bound to SMG-9 and SMG-8

SupramoleculeName: Human SMG-1 kinase bound to SMG-9 and SMG-8 / type: sample / ID: 1000
Oligomeric state: One molecule of SMG-1 binds to one molecule of SMG-9 and one molecule of SMG-8
Number unique components: 3
Molecular weightTheoretical: 580 KDa

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Macromolecule #1: SMG-1

MacromoleculeName: SMG-1 / type: protein_or_peptide / ID: 1 / Name.synonym: SMG-1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 410 KDa
Recombinant expressionOrganism: 293T cells / Recombinant plasmid: pEF_Flag-HA-SBP
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay

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Macromolecule #2: SMG-9

MacromoleculeName: SMG-9 / type: protein_or_peptide / ID: 2 / Name.synonym: SMG-9 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 60 KDa
Recombinant expressionOrganism: 293T Cells / Recombinant plasmid: pSR_Strep-HA
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay

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Macromolecule #3: SMG-8

MacromoleculeName: SMG-8 / type: protein_or_peptide / ID: 3 / Name.synonym: SMG-8 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 110 KDa
Recombinant expressionOrganism: 293T Cells / Recombinant plasmid: pSR_Strep-HA
SequenceGO: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay

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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

BufferpH: 7.5
Details: 10 mM HEPES-KOH at pH7.5, 150 mM NaCl, 20% glycerol, 10 mM MgCl2
StainingType: NEGATIVE
Details: Protein was adsorbed for 1 minute, washed in two water drops and stained with 2% w/v uranyl formate for 1 minute.
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1230
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.9 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry single tilt holder / Specimen holder model: JEOL / Tilt angle max: 20
TemperatureAverage: 294 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 80,000 times magnification
DetailsMicroscope JEOL JEM-1230
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10.5 µm / Details: Minolta Dimage Scan Multi PRO scanner / Bits/pixel: 16
Tilt angle min0

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, Xmipp / Number images used: 13853
DetailsParticles were collected manually.

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