Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and x-ray diffraction to 12.5 A.

The three-dimensional structure of the nicotinic acetylcholine receptor (AChR) from Torpedo californica, crystallized both before and after removal of associated proteins, most notably the main 43-kD cytoskeletal protein that interacts both with AChR and actin, is determined to a resolution of 22 A. This is the first structural analysis where the 43-kD protein has been removed from the sample before crystallization. Thus, it provides the most reliable assessment of what constitutes the structure of the minimal five subunit AChR complex, and, by comparison with the native membrane, of the location of the 43-kD cytoskeletal protein. Image reconstruction of two-dimensional crystals includes information from electron images of up to +/- 52 degrees tilted specimens of latticed AChR. Hybrid density maps that include x-ray diffraction perpendicular to the membrane to 12.5 A resolution were used and eliminate some of the distortions introduced in maps based only on electron microscopic analyses. Comparison of the difference Fourier density maps between AChR with its normal complement of associated proteins, and without them shows that the main density, assigned to the actin-binding 43-kD component is closely associated with the lipid bilayer as well as with the cytoplasmic domain of the AChR. It binds beside the AChR, not beneath it as suggested by others (C. Toyoshima and N. Unwin 1988. Nature [Lond.]. 336:237-240). There is good agreement between the volumes of density for structural components and expected volumes based on their molecular weight. Acetylcholine receptors aggregate in the absence of any cytoskeletal proteins, suggesting that the AChR alone is sufficient to encode and stabilize clustering, and perhaps to do so during synaptogenesis. The main 43-kD component may play a role in location and rate of association of AChR. We show that the disulfide bond that cross-links delta-delta chains of adjacent pentamers in about 80% of AChR, is not required to stabilize the lattice of AChR. Latticed tube structures are stable indefinitely. The lattices described here have 20% less volume of lipid than those originally obtained and characterized by J. Kistler and R. M. Stroud (1981. Proc. Natl. Acad. Sci. USA. 78:3678-3682), or those subsequently characterized by A. Brisson and P. N. T. Unwin (1984. J. Cell Biol. 99:1202-1211) and A. Brisson and P. N. T. Unwin (1985. Nature (Lond.). 315:474-477).