Salk Institute for Biological Studies
Single particle cryo-electron microscopy (cryoEM) is an important component of a structural biologist’s toolkit, as improvements in instrumentation, software, automation, and specimen preparation are making this technique increasingly powerful for the analysis of large (>100 kDa) macromolecules and macromolecular complexes. One of the primary advantages of the methodology is the ability to analyze heterogeneous macromolecular assemblies, i.e. those that exhibit either conformational mobility within distinct regions or compositional heterogeneity exhibited by loosely and sub-stoichiometrically associated components. By employing classification techniques, it is possible to place each individual particle image into one of several, potentially many, groups, according to homogeneity. I will describe recent advances in the use of classification techniques to decipher structural heterogeneity within macromolecular complexes by cryoEM. Using a highly heterogeneous experimental dataset of a 60S ribosomal subunit with several associated non-ribosomal cofactors, I will show how global classification tools can recover numerous conformational and compositional states of mobile components, including subnanometer resolution reconstructions of loosely associated and sub-stoichiometric proteins. I will then zoom in on remaining areas of heterogeneity using a focused classification approach to show how one can deconvolute the mobility of small mobile regions which was not possible using a global classification approach alone, including a single flexible RNA helix. Lastly, I will discuss strategies for improving classification methods and progress toward capturing and deciphering large-scale structural heterogeneity within a single cryoEM data set.