Abstract
With the advent of computationally feasible approaches to maximum-likelihood (ML) image processing for cryo-electron microscopy, these methods have proven particularly useful in the classification of structurally heterogeneous single-particle data. A growing number of experimental studies have applied these algorithms to study macromolecular complexes with a wide range of structural variability, including nonstoichiometric complex formation, large conformational changes, and combinations of both. This chapter aims to share the practical experience that has been gained from the application of these novel approaches. Current insights on how to prepare the data and how to perform two- or three-dimensional classifications are discussed together with the aspects related to high-performance computing. Thereby, this chapter will hopefully be of practical use for those microscopists wishing to apply ML methods in their own investigations.