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- EMDB-2182: Electron microscopy map of SOS1 antiporter -

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Basic information

Entry
Database: EMDB / ID: EMD-2182
TitleElectron microscopy map of SOS1 antiporter
Map dataSOS1 antiporter class 2
Sample
  • Sample: Antiporter SOS1
  • Protein or peptide: Na+/H+ antiporter SOS1
Keywordsmembrane protein / plant salt tolerance / protein structure
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / negative staining / Resolution: 25.9 Å
AuthorsNunez Ramirez R / Sanchez Barrena MJ / Villalta I / Vega JF / Pardo JM / Quintero FJ / Martinez Salazar J / Albert A
CitationJournal: J Mol Biol / Year: 2012
Title: Structural insights on the plant salt-overly-sensitive 1 (SOS1) Na(+)/H(+) antiporter.
Authors: Rafael Núñez-Ramírez / María José Sánchez-Barrena / Irene Villalta / Juan F Vega / Jose M Pardo / Francisco J Quintero / Javier Martinez-Salazar / Armando Albert /
Abstract: The Arabidopsisthaliana Na(+)/H(+) antiporter salt-overly-sensitive 1 (SOS1) is essential to maintain low intracellular levels of toxic Na(+) under salt stress. Available data show that the plant ...The Arabidopsisthaliana Na(+)/H(+) antiporter salt-overly-sensitive 1 (SOS1) is essential to maintain low intracellular levels of toxic Na(+) under salt stress. Available data show that the plant SOS2 protein kinase and its interacting activator, the SOS3 calcium-binding protein, function together in decoding calcium signals elicited by salt stress and regulating the phosphorylation state and the activity of SOS1. Molecular genetic studies have shown that the activation implies a domain reorganization of the antiporter cytosolic moiety, indicating that there is a clear relationship between function and molecular structure of the antiporter. To provide information on this issue, we have carried out in vivo and in vitro studies on the oligomerization state of SOS1. In addition, we have performed electron microscopy and single-particle reconstruction of negatively stained full-length and active SOS1. Our studies show that the protein is a homodimer that contains a membrane domain similar to that found in other antiporters of the family and an elongated, large, and structured cytosolic domain. Both the transmembrane (TM) and cytosolic moieties contribute to the dimerization of the antiporter. The close contacts between the TM and the cytosolic domains provide a link between regulation and transport activity of the antiporter.
History
DepositionAug 24, 2012-
Header (metadata) releaseSep 5, 2012-
Map releaseOct 3, 2012-
UpdateNov 28, 2012-
Current statusNov 28, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.035
  • 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_2182.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSOS1 antiporter class 2
Voxel sizeX=Y=Z: 3.3 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.050022 - 0.22859
Average (Standard dev.)0.00244039 (±0.01969347)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin111
Dimensions808080
Spacing808080
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.33.33.3
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z264.000264.000264.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS111
NC/NR/NS808080
D min/max/mean-0.0500.2290.002

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Supplemental data

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Sample components

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Entire : Antiporter SOS1

EntireName: Antiporter SOS1
Components
  • Sample: Antiporter SOS1
  • Protein or peptide: Na+/H+ antiporter SOS1

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Supramolecule #1000: Antiporter SOS1

SupramoleculeName: Antiporter SOS1 / type: sample / ID: 1000
Details: This sample is a membrane protein and is purified with lipids and detergents surrounding the membrane domain
Oligomeric state: Dimer / Number unique components: 1
Molecular weightExperimental: 360 KDa / Theoretical: 254 KDa
Method: Gel filtration, Electron microscopy image analysis, Dynamic light scattering

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Macromolecule #1: Na+/H+ antiporter SOS1

MacromoleculeName: Na+/H+ antiporter SOS1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Arabidopsis thaliana (thale cress) / Location in cell: Plasma membrane
Molecular weightTheoretical: 127 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: pYPGE15

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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: 8.5
Details: 50 mM phosphate buffer pH 8.5, 300 mM NaCl, 10% glycerol, 0.05% DDM, 250 mM imidazole
StainingType: NEGATIVE
Details: Purified SOS1 was adsorbed to glow-discharged carbon coated grids and stained with 2% uranyl formate
GridDetails: 400 mesh carbon coated copper grid, glow discharged in moderate vacuum
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 20000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 20000
Sample stageSpecimen holder model: JEOL
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120.000-150.000 times magnification and confirmed by inspection of the Fourier transform
DateNov 1, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ORIUS SC200 (2k x 2k) / Number real images: 90 / Average electron dose: 15 e/Å2 / Bits/pixel: 14
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Each micrograph. Estimation using CTFFIND3 and correction using Bsoft
Final two d classificationNumber classes: 96
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: XMIPP / Number images used: 6300
DetailsSingle images of SOS1 were manually extracted using EMAN. 2D reference-free classification, 2D averaging, 3D classification and reconstruction were performed using maximum-likelihood methods implemented in XMIPP package.

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