Mass Mapping of Macromolecular Assemblies

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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 characterize the composition of post-synaptic densities (PSDs) that are isolated from rat forebrain, frozen rapidly, and supported on thin carbon substrates. The PSD is a complex molecular machine, which underlies the post-synaptic membrane and is responsible for organizing receptors and signaling molecules at the post-synaptic active zone. It is found that PSDs become thicker upon exposure of the neurons to ischemia as well as other excitatory conditions and it is known that the enzyme CaMKII contributes to this thickening. We have used STEM mass mapping to estimate the number of CaMKII molecules that are added to the PSD under excitatory conditions. Knowledge of the total mass of the PSD opens the way to a quantitative and stochiometric analysis of functional changes in the protein composition of PSDs