Studying the structure of microtubules by electron microscopy

Although the structures of individual proteins and moderately sized complexes of proteins may be investigated by X-ray crystallography, the interaction between a long polymer, such as a microtubule, and other protein molecules, such as the motor domain of kinesin, need to be studied by electron microscopy. We have used electron cryo-microscopy and image analysis to study the structures of microtubules with and without bound kinesin motor domains and the changes that take place when the motor domains are in different nucleotide states. Among the microtubules that assemble from pure tubulin, we select a minor subpopulation that has perfect helical symmetry, which are the best for three-dimensional reconstruction. Gold labeling can be used to mark the positions of certain regions of protein sequence.