Quantitation of endosome morphology and vesicle budding profiles. Quantitation of the mean number of ILV budding profiles observed per MVB (A) versus the frequency distribution of the number of ILV budding profiles observed per MVB (B) observed in tomograms. Quantitation of the relative percentage of limiting versus lumenal membrane surface areas (C) and the mean ILV diameters (D) observed in tomograms. (E) The percentage of the limiting membrane incorporated into ILV budding profiles was measured for each strain. Note that no ILV budding profiles were seen for snf7Δ cells, but five freely detached ILVs were observed at the periphery of a class E compartment in a single tomogram of snf7Δ cells (Fig. S5); the rarity of this occurrence is reflected by the observation that the membrane surface area of these ILVs comprised 0.27% of the total endosomal membrane surface area in this strain. Tomograms for 12, 14, and 17 MVBs were prepared and modeled for wild-type cells, cells overexpressing the Bro1 domain, and cells overexpressing full-length Bro1, respectively. Four individual tomograms were prepared and modeled for snf7^L231A/L234A cells, which contained a total of 33 MVB, VTE, and cisternal structures. Three individual tomograms were generated and modeled for snf7Δ cells, each containing E compartments that were composed of multiple cisternal stacks and occasionally also spherical endosomal membranes. Methods used for the preparation of the tomograms that were used to generate the measurements shown in this experiment are described in detail in the Materials and methods section.