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- EMDB-2313: 3D reconstruction of Mechanosensitive Channel Candidate MCA2 -

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Basic information

Entry
Database: EMDB / ID: EMD-2313
Title3D reconstruction of Mechanosensitive Channel Candidate MCA2
Map dataReconstruction of MCA2 in detergent solubilized state from Zernike Phase Contrast cryoEM images
Sample
  • Sample: MCA2
  • Protein or peptide: Mid1-complementing activity 2
KeywordsMechanosensitive channel candidate / membrane protein / Calcium uptake
Function / homology
Function and homology information


post-embryonic root development / calcium channel activity / membrane => GO:0016020 / cell surface receptor signaling pathway / plasma membrane
Similarity search - Function
PLAC8 motif-containing protein / PLAC8 motif-containing protein / PLAC8 family / Adaptor protein Cbl, N-terminal domain superfamily
Similarity search - Domain/homology
Protein MID1-COMPLEMENTING ACTIVITY 2
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 26.0 Å
AuthorsShigematsu H / Iida K / Nakano M / Chaudhuri P / Iida H / Nagayama K
CitationJournal: PLoS One / Year: 2014
Title: Structural characterization of the mechanosensitive channel candidate MCA2 from Arabidopsis thaliana.
Authors: Hideki Shigematsu / Kazuko Iida / Masataka Nakano / Pratima Chaudhuri / Hidetoshi Iida / Kuniaki Nagayama /
Abstract: Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca²⁺-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from ...Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca²⁺-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca²⁺ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures. Because of the abundance of protein in these cells, MCA2 was chosen for purification. Purified MCA2 in a detergent-solubilized state formed a tetramer, which was confirmed by chemical cross-linking. Single-particle analysis of cryo-electron microscope images was performed to depict the overall shape of the purified protein. The three-dimensional structure of MCA2 was reconstructed at a resolution of 26 Å from 5,500 particles and appears to comprise a small transmembrane region and large cytoplasmic region.
History
DepositionFeb 12, 2013-
Header (metadata) releaseMar 6, 2013-
Map releaseFeb 5, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.89
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.89
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2313-For...

Downloads & links

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Map

FileDownload / File: emd_2313.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of MCA2 in detergent solubilized state from Zernike Phase Contrast cryoEM images
Voxel sizeX=Y=Z: 3 Å
Density
Contour LevelBy AUTHOR: 2.89 / Movie #1: 2.89
Minimum - Maximum-3.86343384 - 10.47515106
Average (Standard dev.)0.0 (±0.89218032)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-13-13-13
Dimensions808080
Spacing808080
CellA=B=C: 240.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z240.000240.000240.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-13-13-13
NC/NR/NS808080
D min/max/mean-3.86310.4750.000

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Supplemental data

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Sample components

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Entire : MCA2

EntireName: MCA2
Components
  • Sample: MCA2
  • Protein or peptide: Mid1-complementing activity 2

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Supramolecule #1000: MCA2

SupramoleculeName: MCA2 / type: sample / ID: 1000
Details: The sample was purified with size-exclusion chromatography finally in the center of peak fraction
Oligomeric state: homotetramer / Number unique components: 1
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Mid1-complementing activity 2

MacromoleculeName: Mid1-complementing activity 2 / type: protein_or_peptide / ID: 1 / Name.synonym: MCA2
Details: recombinantly expressed in Sf9 and purified under ammonium perfluorooctanoate.
Number of copies: 1 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Arabidopsis thaliana (thale cress) / synonym: Arabidopsis / Location in cell: Plasma membrane
Molecular weightTheoretical: 200 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant cell: Sf9 / Recombinant plasmid: pFastBac1
SequenceUniProtKB: Protein MID1-COMPLEMENTING ACTIVITY 2 / InterPro: PLAC8 motif-containing protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 8 / Details: PBS, 4% ammonium perfluorooctanoate
GridDetails: thin carbon over the Quantifoil R1.2/1.3, glow discharged in air
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeOTHER
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.7 mm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: JEOL / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Liquid Helium cooled stage maintained around 55 K
Specimen holder model: JEOL
TemperatureMin: 45 K / Max: 60 K / Average: 55 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateMay 10, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (2k x 2k) / Digitization - Sampling interval: 30 µm / Number real images: 50 / Average electron dose: 20 e/Å2 / Bits/pixel: 8

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Image processing

Final two d classificationNumber classes: 133
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 5700
DetailsParticles were selected using boxer.

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