Three different approaches for calculating the three-dimensional structure of microtubules decorated with kinesin...

We have used three different electron microscopy approaches to calculate three-dimensional maps of tubulin assemblies decorated with the motor domain of kinesin. The approaches used were: (1) Tilt series reconstruction of negatively stained tubulin sheets. (2) Back-projection reconstruction of microtubules in ice. (3) Helical reconstruction of microtubules in ice. The calculated maps show the overall configuration of the protofilaments and the interactions between the motor and the protofilaments at a resolution of 2-4 nm. The three methods revealed a similar binding configuration of the kinesin motor domain to the protofilament. We also found that seams can be present in potentially helical microtubules, limiting the use of helical reconstruction algorithms. Advantages and disadvantages of each of the three approaches are discussed.