Mass Mapping Of Macromolecular Assemblies

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Abstract

Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. Macromolecules are adsorbed onto a thin support film and a nanometer-sized electron probe is scanned across the specimen while the elastic-scattering signal is collected. The resulting digital image intensity is proportional to the local mass density of the specimen. Images can be recorded at low electron dose without significant radiation damage to the structures of interest. We have used this approach to investigate the composition of Spiroplasma citri, a simple bacterium, which is of interest because of its motility-producing contractile cytoskeleton, which also functions as a linear motor. This motor may serve as a model system for understanding the function of the cytoskeleton in other cells. In addition, to determining the organization of the contractile linear motor, we are now characterizing the membrane structure and determining Spiroplasma's composition in terms of protein, lipid and nucleic acid content. It is expected that the results will provide some information about the minimum requirements to sustain the cell's free-living state.