ELECTRON MICROSCOPY OF CHAPERONINS

Publisher Summary The chapter presents a study related to electron microscopy of chaperonins. The chapter discusses several electron microscopy (EM) and image-processing methods that are useful for the study of chaperonin and the different kinds of information that they provide. EM plays an important role in understanding chaperonin structure and function. The large size and high symmetry of chaperonin oligomers make it possible to deduce detailed two-dimensional (2D) and three-dimensional (3-D) structural information, and the time resolution of cryo-EM in particular makes it a powerful technique for dynamic studies of chaperonin assisted protein folding. There are three commonly used negative stains: uranyl acetate (UA), phosphotungstic acid (PTA), and ammonium molybdate (AM), normally used at 2% (w/v). Among them UA gives the best contrast and shows good results with many, but not all, protein specimens. EM studies have made significant contributions to the understanding of chaperonin structure and function, aided by the high symmetry and the highly cooperative conformational changes within the rings of subunits. Negative cooperativity between the two rings of GroEL is demonstrated by its preference for single-sided binding of cpnl0 and substrates. Even with crystal structures available for Escherichia coli GroEL, GroES, and their complex, cryo-EM, reconstructions can be combined with atomic structure information to study transient conformational changes and characterize complexes with folding substrates.