Gating mechanism of the acetylcholine receptor ion channel

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The ultimate goal of this joint research is to elucidate the gating mechanism of the acetylcholine receptor by studying the three-dimesional structure by cryo-electron microscopy. The specific aims were to analyze the tubular crystals at a higher resolution and to visualise the three-dimensional structure of the receptor in different physiological states. To do so, I made three visits and Dr Unwin made one for the the period supported.The first one includes the development of the programs for determining the CTF parameters precisely from electron micrographs. We have developed a set of programs running under the X-window and ported them to the computers at the MRC laboratory. These programs have proven to be very useful and robust. Therefore, we have written a paper, with the help of Dr Unwin, and submitted to Ultramicroscopy.The other aim was to develop programs for correcting small distortions in crystal lattice of a tubular crystal. We have developed a method to correct them in real space and Unwin's group have explored the way in which the helix axis position was refined in reciprocal space for 3 segments constituting one repeat. We have been trying to combine both ways to provide better correction scheme.For time-resolving experiments, different methods have been pursued. We use flash photolysis of caged-compounds and Unwin uses spraying of agonist (or ligand). Flash photolysis is superior in terms of timeresolution but it cannot neglect the problem of temperature rise. Hence we are implementing the spraying apparatus in our machine. To acquire enough experience with spary freezing I prepared many specimens and examined them during my stay in England. One practical problem was that the numbers of tubes found on microscope grids were rather small. This problem was solved by concentrating the specimen by powder of polyethylen glycol.