FIG. 3.
(a to c) Negatively stained electron micrographs obtained from a Tween 80 extract of cells expressing Tsr. The extract preserved the overall appearance of the membrane morphologies in the tomogram, and the images show in more detail the organization of the zippered structures in cross section (a) and top view (b), as well as rounded vesicular structures (c). Scale bars = 50 nm. (d) Molecular model for packing in the zippered regions based on the atomic model for the structure of Tsr shown in Fig. 1a and the electron crystallographic analysis of the crystalline regions (32). The packing in the crystalline areas of the membrane (b) roughly corresponds to a lattice with the following constants: a, ∼75 Å; b, ∼75 Å; and γ, ∼60°. The cross-sectional area of each unit in the crystal is ∼5,000 Å2, which can accommodate at most three Tsr dimers, based on the presence of four helices per Tsr monomer at the wider, periplasmic end of the receptor (20) and the known values of about 180 Å2 that characterize the cross-sectional areas of helices in well-packed two-dimensional crystalline arrays (12). The array shown is one of many possible arrangements of the three dimers and is presented primarily to provide an indication of the density of receptor packing in the membrane. The white regions at the center of the zippered structure in panel a are interpreted as high-density regions where the cytoplasmic ends of the Tsr trimer are presumed to be interdigitated. The depth of the interdigitation is consistent with the proportional width of this region in the micrograph in panel a. The average internal spacing of the zippered structures in tomographic slices such as the slice shown in Fig. 2c is 31.5 + 2.5 nm (averaged over 26 separate measurements). This compares well with the value for the same internal spacing in these negatively stained specimens (31.3 ± 0.6 nm [averaged over 10 measurements], as reported by Weis et al. [32]). (e) Molecular model for packing in the vesicular regions (sectional view). The outer white ring of the circular structure in panel c is interpreted as the surface of the membrane region enclosing the volume, and the less clear inner ring is interpreted as a high-density region where the cytoplasmic ends of Tsr come together.