+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2313 | |||||||||
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Title | 3D reconstruction of Mechanosensitive Channel Candidate MCA2 | |||||||||
Map data | Reconstruction of MCA2 in detergent solubilized state from Zernike Phase Contrast cryoEM images | |||||||||
Sample |
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Keywords | Mechanosensitive 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 | |||||||||
Biological species | Arabidopsis thaliana (thale cress) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 26.0 Å | |||||||||
Authors | Shigematsu H / Iida K / Nakano M / Chaudhuri P / Iida H / Nagayama K | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2313.map.gz | 1.3 MB | EMDB map data format | |
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Header (meta data) | emd-2313-v30.xml emd-2313.xml | 10 KB 10 KB | Display Display | EMDB header |
Images | EMD-2313-For_EMDB.tif | 950 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2313 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2313 | HTTPS FTP |
-Validation report
Summary document | emd_2313_validation.pdf.gz | 189.6 KB | Display | EMDB validaton report |
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Full document | emd_2313_full_validation.pdf.gz | 188.7 KB | Display | |
Data in XML | emd_2313_validation.xml.gz | 4.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2313 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2313 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2313.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of MCA2 in detergent solubilized state from Zernike Phase Contrast cryoEM images | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : MCA2
Entire | Name: MCA2 |
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Components |
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-Supramolecule #1000: MCA2
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 200 KDa |
-Macromolecule #1: Mid1-complementing activity 2
Macromolecule | Name: 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 |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) / synonym: Arabidopsis / Location in cell: Plasma membrane |
Molecular weight | Theoretical: 200 KDa |
Recombinant expression | Organism: unidentified baculovirus / Recombinant cell: Sf9 / Recombinant plasmid: pFastBac1 |
Sequence | UniProtKB: Protein MID1-COMPLEMENTING ACTIVITY 2 / InterPro: PLAC8 motif-containing protein |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.05 mg/mL |
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Buffer | pH: 8 / Details: PBS, 4% ammonium perfluorooctanoate |
Grid | Details: thin carbon over the Quantifoil R1.2/1.3, glow discharged in air |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | OTHER |
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Temperature | Min: 45 K / Max: 60 K / Average: 55 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification |
Specialist optics | Energy filter - Name: JEOL / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Date | May 10, 2008 |
Image recording | Category: 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 3.7 mm / Nominal magnification: 60000 |
Sample stage | Specimen holder: Liquid Helium cooled stage maintained around 55 K Specimen holder model: JEOL |
-Image processing
Details | Particles were selected using boxer. |
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Final reconstruction | Applied 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 |
Final two d classification | Number classes: 133 |