Abstract
This chapter focuses on electron microscopy-based methods to assess the structural and functional aspects of kinesins and dyneins in complex with microtubules. It describes two fundamentally different approaches for the study of motor–microtubule complex structure in three dimensions (3D). Both methods have been applied very successfully in the past, but further refinement is needed to generate a comprehensive model of the intracellular transport machinery at near-atomic resolution. The more established and older 3D reconstruction methods apply averaging of image elements to regenerate a 3D data set from many different projections of apparently identical particles. More recently advances in image recording technology and the availability of dramatically increased computing power has initiated new approaches that now apply tomographic 3D reconstruction to essentially any type of molecular and cellular structure without the need for averaging. The chapter describes 3D cryoelectron microscopy (cryo-EM) methods that create structural data in a resolution range of 1 to 4 nm, which is suitable for a detailed interpretation in conjunction with X-ray and electron crystallography or nuclear magnetic resonance (NMR) spectroscopy.