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| Title: | Structure and gating mechanism of the acetylcholine receptor pore. |
| Authors: | A.Miyazawa,Y.Fujiyoshi,N.Unwin |
| Sample: | Crystalline postsynaptic membrane from Torpedo marmorata
electric organ |
| Aggregation state: | Helical (4.0 angstroms resolution) |
 Latest update: | 2011-05-26 |
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| Sample |
| Sample name:
| Crystalline postsynaptic membrane from Torpedo marmorata
electric organ |
| Oligomeric state:
| The acetylcholine receptors are hetero-pentamers
composed of 2 alpha 1 beta 1 gamma and 1 delta subunit |
| Components:
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| Experiment |
| Sample preparation: |
| pH | Sample conc. | Details | Staining | Sample support details |
| 6.8 | mg/mL | 100mM sodium cacodylate, 1mM CaCl2 | 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. |
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| Vitrification: |
| Cryogen name | Humidity | Temp. | Instr. | Method | Time resolved | Details |
| ETHANE | 90% | 100 K | HOMEMADE PLUNGER | 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. | ms | 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. |
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| Imaging: |
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| Processing |
| Software: | In-house software based on MRC system |
| CTF correction: | Measurement of positions of Thon rings from
area of tube that was processed |
| Resolution by author: | 4.0 Å
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| Resolution method: | FSC at 0.5 cut-off |
| Processing details: | 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. |
| Unit cell: |
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| Scanned images: |
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| Fitting: |
| PDB | Protocol | Target crit. | Software | B value | Fitting space | PDB chain | Details |
| 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. |
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