|Entry||Database: EMDB / ID: 2071|
|Title||Gating movement in acetylcholine receptor analysed by time-resolved electron cryo-microscopy|
|Keywords||acetylcholine receptor / freeze-trapping / asymmetric gating / allosteric mechanism|
|Sample||nicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata|
|Source||Torpedo marmorata / fish / marbled electric ray / image: Torpedo californica|
|Map data||Density map of acetylcholine receptor|
|Method||helical reconstruction, at 6.2 Å resolution|
|Authors||Unwin N / Fujiyoshi Y|
|Citation||J. Mol. Biol., 2012, 422, 617-634|
|Validation Report||PDB-ID: 4aq5|
SummaryFull reportAbout validation report
|Date||Deposition: Apr 12, 2012 / Header (metadata) release: Apr 17, 2012 / Map release: Aug 1, 2012 / Last update: Sep 26, 2012|
Downloads & links
|File||emd_2071.map.gz (map file in CCP4 format, 10753 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 1 Å|
CCP4 map header:
-Entire nicotinic acetylcholine receptor in native postsynaptic membrane ...
|Entire||Name: nicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata|
Number of components: 1 / Oligomeric State: 5 subunits
|Mass||Theoretical: 300 kDa / Experimental: 300 kDa|
Measured by: molecular weight based on amino acid sequence data and attached sugars
-Component #1: protein, nicotinic acetylcholine receptor
|Protein||Name: nicotinic acetylcholine receptor / a.k.a: nicotinic receptor / Oligomeric Details: pentamer|
Details: Protein is embedded in postsynaptic membrane isolated from Torpedo marmorata electric organ
Recombinant expression: No
|Mass||Theoretical: 300 kDa / Experimental: 300 kDa|
|Source||Species: Torpedo marmorata / fish / marbled electric ray / image: Torpedo californica|
|Source (natural)||Organelle: plasma membrane / Location in cell: plasma membrane / Cell: electrocyte cells / Organ or tissue: electric organ|
|Specimen state||helical array|
|Helical parameters||Hand: RIGHT HANDED|
|Crystal grow details||Tubular membrane crystals of acetylcholine receptors grow spontaneously from isolated postsynaptic membranes when incubated in low salt buffer at 17 degrees C for two weeks|
|Sample solution||Buffer solution: 100 mM sodium cacodylate, 1 mM calcium chloride|
|Support film||300 mesh copper grid with pre-irradiated thick holey carbon support, glow discharged in amylamine atmosphere|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 85 %|
Method: Blot until applied droplet loses contact with filter paper (indicated by loss of transparency; typically 6s)
Time resolved state: Vitrified within 10ms of exposure to acetylcholine (applied as the grid is being plunged,using a fine, focussed spray positioned about 1cm above the ethane surface)
Details: Vitrification carried out at an ambient temperature of 8 degrees C
-Electron microscopy imaging
|Imaging||Microscope: JEOL 3000SFF / Date: Nov 1, 2005|
Details: Standard low dose imaging of specimens over holes in the carbon support film
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 40000 X (nominal), 38500 X (calibrated)|
Astigmatism: Objective lens astigmatism was corrected based on appearance of carbon film at 250,000 times magnification
Cs: 1.6 mm / Imaging mode: BRIGHT FIELD / Defocus: 900 - 2000 nm
|Specimen Holder||Holder: Top-entry holder for liquid helium cooled stage (the temperature of the specimen in this holder is usually at 4K)|
Model: OTHER / Temperature: 10 K ( 10 - 20 K)
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 111 / Scanner: OTHER / Sampling size: 2.5 microns / Bit depth: 16 / OD range: 1|
Details: All images recorded on film, developed in Kodak d19 developer
|Processing||Method: helical reconstruction|
Details: Alignment and distortion correction of each tube image was done using a segmental Fourier-Bessel method (Beroukhim & Unwin (1997) Ultramicroscopy, 70:57-81) with 50% overlap between successive segments
|3D reconstruction||Resolution method: FSC 0.5|
Details: Final maps were calculated from 111 tube images(closed class) and 123 tube images (open class)
Software: MRC, and, own, programs / Algorithm: Standard Fourier-Bessel synthesis / CTF correction: Each tube image / Resolution: 6.2 Å
-Atomic model buiding
|Modeling #1||Software: DireX / Refinement protocol: flexible / Refinement space: REAL|
Details: Protocol: Maximisation of correlation between experimental densities and atomic model, using a deformable elastic network algorithm. Identical refinement procedures were applied to both density maps. The fits were validated by applying the same refinement procedures to independent density maps calculated from half-datasets
Input PDB model: 2BG9
Chain ID: A, B, C, D, E
-Oct 4, 2017. Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
- Jacques Dubochet (University of Lausanne, Switzerland) is a pioneer of ice-embedding method of EM specimen (as known as cryo-EM), Most of 3DEM structures in EMDB and PDB are obtained using his method.
- Joachim Frank (Columbia University, New York, USA) is a pioneer of single particle reconstruction, which is the most used reconstruction method for 3DEM structures in EMDB and EM entries in PDB. And also, he is a develper of Spider, which is one of the most famous software in this field, and is used for some EM Navigor data (e.g. map projection/slice images).
- Richard Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) was determined the first biomolecule structure by EM. The first EM entry in PDB, PDB-1brd is determinedby him.
External links: The 2017 Nobel Prize in Chemistry - Press Release
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