Many membrane proteins, soluble proteins and artificial protein-lipid complexes have a preference to assemble into tubular crystals under near-physiological conditions. Such specimens are ideal for structural and functional studies by electron microscopy, as was demonstrated by our recent study leading to a refined atomic model of the nicotinic acetylcholine (ACh) receptor. The purpose of the proposed research is: (a) to complement that analysis of the receptor in the closed-channel form by determining the high resolution structure of the receptor in the open-channel form, and hence obtain an understanding of the structural basis of gating in atomic-scale detail;(b) to improve the methodology for solving structures from tubular crystals so that near-atomic models of protein assemblies can be derived from all kinds of tubular crystal quickly and with minimum effort. A rapid spray-freezing technique will be used to achieve an appropriate brief (~5 ms) reaction time and trap the channels in the open state, before significant desensitization can take place. The imaging will be conducted at liquid helium temperatures to minimize radiation damage and to optimize electron-optical performance. The image processing will make use of previously developed and new routines, which will be automated in collaboration with colleagues working on related specimens at Scripps;this will enable rapid screening for the best images, which is the main step at present limiting the rate of high resolution structure determination. An understanding of the gating mechanism of the ACh receptor (and hence of other transmitter-gated ion channels) is relevant to many kinds of neurological disorder, because it is the fundamental process underlying all fast synaptic communication in the nervous system. The cryo-electron crystallographic approach we use and are developing, is important because it is the most powerful method available to analyze the structures of membrane proteins in their native lipid environment and under near-physiological ionic conditions.
