Methods in Molecular Medicine, Cardiovascular Disease

Cryo-electron microscopy (cryo-EM) has a wide application to the analysis of biological structures. The focus of this chapter is the use of cryo-EM and associated techniques to examine the three dimensional (3D) structure of single proteins and multi-component macromolecular assemblies. Data evolving from these methods, pertaining to the quaternary organisation of proteins and protein-protein interactions, bridges an important gap between linear, genomic, information towards understanding physiological function. This chapter provides methods for examining two-dimensional crystalline arrays of proteins as well as single randomly orientated proteins. It is significant that single particle analysis of electron microscopy images has provided the only 3D data to-date for the two principal components of muscle excitation contraction coupling in the heart, namely the L-type calcium channels and the ryanodine receptors. Approaches are described for identifying the extracellular and intracellular domains of the 3D structure of the L-type voltage gated calcium channel and also incorporates general details for labeling and visualising His-tagged proteins by electron microscopial methods.