MASS MAPPING OF MACROMOLECULE ASSEMBLIES

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The VG Microscopes HB501 field-emission STEM has been used to record low- dose digital annular dark-field mass maps of isolated, rapidly frozen protein macromolecular assemblies. Data were recorded from microtubules complexed with the cytoplasmic motor protein, kinesin. First, the molecular weight of free kinesin molecules (approximately 380 +/- 15 kDa) was established by calibrating the digital micrographs with tobacco mosaic virus particles and with the 13-protofilament microtubules. Then, the masses of the attached kinesins were measured in order to characterize how they interact with the microtubule. The results show the first direct evidence of cross-bridging of microtubules by single kinesin molecules, and indicate that kinesins bind to microtubules not only by their heavy chains in the head domain, but also by a light-chain binding site in the foot. Preliminary molecular weight data were also obtained from an important phosphorylation protein, CAM kinase II, found in the postsynaptic density of the brain.