Three-dimensional Image Analysis of Tubular Crystals of Membrane Proteins in Ice

Frozen-hydrated electron microscopy is a powerful method that allows us to preserve biological macromolecules in physiological ionic conditions and obtain the density (Coulomb potential) maps directly. Changes in quarternary structure in different conditions have been demonstrated using this technique for the gap junction (1) and the nicotinic acetylcholine receptor (2). Since the image contrast originates from the difference in density between the specimen and ice, the method has a considerable advantage over conventional methods in resolving structure inside the lipid bilayer, especially when molecules are arranged in a helical array to form tubular crystals. The molecules in a helical array present many different views to the incident electron beam, hence the electron micrograph actually contains a large amount of three-dimensional information. By using helical image analysis, it is possible to obtain a three-dimensional image from a single micrograph in many instances. Furthermore, the data is complete: There is no "missing cone" problem arising from a limited angle of tilt; the resolution in the reconstructed image is isotropic. The mean (radial) density distribution is available from equatorial data; thus the value in the map is an absolute measure of the density.We have been analysing tubular crystals of the nicotinic acetylcholine receptor from electric ray and the calcium ATPase from rabbit sarcoplasmic reticulum. Helical image analysis at 17 Å resolution of the narrow tubes embedded in ice has allowed two leaflets of the lipid bilayer to be resolved clearly (2). The lipid bilayer was not resolved in previous tilt reconstructions using flattened tubes at a similar (in plane) resolution and up to 60 degrees tilt (3).