The scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold-field emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. We have shown that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can provide accurate STEM mass measurements, which can elucidate the organization of a wide variety of macromolecular assemblies, and even entire bacterial cells. A particularly important application of the STEM mass mapping technique is to study the organization of disease-related filamentous structures of which amyloid fibrils implicated in Alzheimers disease constitute an important and well-studied class. In general terms, amyloid fibrils consist of a cross-beta sheet structure in which beta-strand segments are aligned perpendicular to the fibril long axis and connected by intermolecular hydrogen bonds with a cross-beta repeat distance of 0.48 nm. The packing of at least two beta-sheets is usually necessary to form a single Alzheimers amyloid protofilament unit. In turn, variations in proto filament number and arrangement contribute to the substantial polymorphism characteristic of amyloid fi brils. Measurements of mass per length (MPL) in the STEM are unique in that they yield the number of peptide molecules per cross-beta repeat on individual fibrils. When combined with information obtained from other structural techniques, this analysis assists in the generation of comprehensive structural models for the assembly of single proto filament subunits into fibril polymorphs. To elucidate how the components of an extremely simple cell are spatially organized, STEM mass mapping has been applied to analyze the molecular inventory of Spiroplasma melliferum, a wall-less free-living bacterium with an exceedingly small genome and dynamic helical geometry. Together with other biophysical measurements, STEM determination of the mass-per-length of whole Spiroplasma, the mass-per-length of the cell's cytoskelatal motor assemblies, and the mass-per-area of its membrane fractions, provide a general framework for a minimal inventory and arrangement of major cellular components that are needed for a minimal viable cell. These local data were fit into whole-cell geometric parameters determined by a variety of light microscopy modalities. Hydrodynamic data obtained by analytical ultracentrifugation allowed computation of the hydration state of whole living cells, for which the relative amounts of protein, lipid, carbohydrate, DNA, and RNA were also estimated analytically. Finally, ribosome and RNA content, genome size and gene expression were also estimated (using stereology, spectroscopy and 2D-gel analysis, respectively). Taken together, the results provide a general framework for a minimal inventory and arrangement of the major cellular components needed by Spiroplasma to support its living state. We expect that the resulting model will prove useful for understanding other cells types.
