2003-04-09 2005-04-09 false A.Miyazawa,Y.Fujiyoshi,N.Unwin Structure and gating mechanism of the acetylcholine receptor pore. Nature 424 949 955 2003 emd_1044.map float (32-bit) 128 128 55 0 0 52 127 127 106 128 128 168 128.000 128.000 168.000 90.000 90.000 90.000 X Y Z -2.23608 8.47376 0.75092 0.00000 1
166160.lg1 // Created by IMAGIC: CCP4 image= achr.map04-04-20 12:36:09 //
1.0 1.0 1.0 The map is of the membrane-spanning domain of the nicotinic acetylcholine receptor in the closed state, viewed from the synaptic cleft. The arrangement of subunits around the central axis, clockwise beginning from the bottom (closest to 0 on the y-axis) ia alpha, gamma, beta, delta. The Fourier terms were derived from tubular crystals having helical symmetry. They are of higher quality along the meridional (y-axis) direction than the equatorial direction (where the diffraction is weaker and there is additional noise associated with layer-line overlap. This has resulted in some asymmetry in the map, with the best direction being along the axis of the tube (y-axis). The map was obtained by averaging data from four helical families in real space, weighting each family approximately according to the number of receptors analysed. The actual weights were: 0.70 (-16,6); 0.30 (-15,7); 0.30 (-17,5); 0.25 (-18,6). As explained in the Reference, the dominating low resolution terms were weakened by subtracting a map of the structure with terms extending to only 15 Angstroms. THe weight used for the subtraction map was -0.88. The terms along the equator have also been included with a weight of 0.04, so that the densities corresponding to the alpha-helical segments are represented at about the same level throughout the thickness of the bilayer.
FSC for accompanying map.
emd_1044_fsc.xml Crystalline postsynaptic membrane from Torpedo marmorata electric organ helical The acetylcholine receptors are hetero-pentamers composed of 2 alpha 1 beta 1 gamma and 1 delta subunit 2 protein acetylcholine receptor 290
This is the MW of the glycosylated protein. The protein itself accounts for 258kD
pentamer false electric organ plasma membrane cellular-component postsynaptic membrane lipids false electric organ plasma membrane ETHANE 90 100 Home-built model The grid was first glow-discharged in the presence of amyl amine. The specimen was applied to the carbon-film side in 4.2ul droplets. Blotting was done from the other side, removing the filter paper and plunging as soon as the paper and grid were observed to lose water-contact with each other - typically after 6 seconds.
The plunging apparatus was contained in a bench-top fridge having a window made in the door. Wet air was continually bubbled into the fridge, which was maintained at 4-8 deg. centigrade.
 JEOL KYOTO-3000SFF top-entry OTHER 300 FLOOD BEAM BRIGHT FIELD 1.3 correction on carbon film at 250,000 800 1800 40000 36800 FIELD EMISSION GUN 20 4.2 4.2 4.2 Kodak SO-163 film 359 5 1 10
Scanning done with a point-source, flat-bed Joyce-Loebl microdensitometer, modified in-house
OTHER 1oed
Interpretation of the experimental density map and model building into the densities were performed using O. The helical segments were fitted individually, using the protruding regions along the helical densities to identify the largest side chains. This allowed tentative assignments to be made of each amino acid according to the sequence, both along the helices and along the short connecting loops. These assignments were then validated for each subunit by checking their consistency with residues in equivalent positions around the pentamer.
100mM sodium cacodylate, 1mM CaCl2
6.8 no stains or fixatives used holey carbon film made over 300 mesh copper grids. To minimise beam movement at the 4K imaging temperature, it was essential that the carbon films had a high electrical conductivity - achieved by evaporation of carbon in a high vacuum and pre-irradiation of the grids. In-house software based on MRC system Measurement of positions of Thon rings from area of tube that was processed 4.0 FSC at 0.5 cut-off
Layer-line data were collected from 4 helical families of tubes - (-16,6),(-15,7),(-17,5),(-18,6) - after dividing the tubes into short segments to correct for distortions. The maps calculated from each of the families were then averaged in real space to derive the final three-dimensional densities.
The specimens were tubular crystals forming a range of helical families, with the receptors in each case being organised on a p2 surface lattice.