REL PDBe PDBe PDBe 2003-04-03 2003-04-09 2005-04-09 2014-06-18 Structure and gating mechanism of the acetylcholine receptor pore. Miyazawa A Fujiyoshi Y Unwin N Miyazawa A Fujiyoshi Y Unwin N Structure and gating mechanism of the acetylcholine receptor pore. NATURE 423 949 955 2003 12827192 doi:10.1038/nature01748 1oed FULLOVERLAP Crystalline postsynaptic membrane from Torpedo marmorata electric organ Crystalline postsynaptic membrane from Torpedo marmorata electric organ The acetylcholine receptors are hetero-pentamers composed of 2 alpha 1 beta 1 gamma and 1 delta subunit 2 postsynaptic membrane lipids false Torpedo marmorata Torpedo marmorata electric organ plasma membrane acetylcholine receptor Torpedo marmorata marbled electric ray electric organ plasma membrane 0.290
This is the MW of the glycosylated protein. The protein itself accounts for 258kD
pentamer false helical filament 6.8
100mM sodium cacodylate, 1mM CaCl2
 NEGATIVE
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.
 ETHANE 90 100 HOMEMADE PLUNGER
Vitrification instrument: Home-built model. 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.
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. JEOL KYOTO-3000SFF FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 1.3 0.8 1.8 40000.0 36800.0 OTHER 4.2 4.2 4.2 correction on carbon film at 250,000 KODAK SO-163 FILM OTHER 5 359 20
Scanning done with a point-source, flat-bed Joyce-Loebl microdensitometer, modified in-house
1.0 10. top-entry
The specimens were tubular crystals forming a range of helical families, with the receptors in each case being organised on a p2 surface lattice.
4.0 FSC 0.5 CUT-OFF In-house software based on MRC system
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.
Measurement of positions of Thon rings from area of tube that was processed
 emd_1044.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 55 0 0 52 128 128 55 128 128 168 90 90 90 Y X Z -2.43914 6.31988 -0.000000169242 1.0 1 1 1 2.18 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.
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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.
 emd_1044_fsc.xml