In Situ Studies of Mitochondrial Translation by Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) enables the three-dimensional (3D) visualization of macromolecular complexes in their native environment (in situ). The ability to visualize macromolecules in situ is in particular advantageous for complex, membrane-associated processes, such as mitochondrial translation. Mitochondrial translation occurs almost exclusively associated with the inner mitochondrial membrane, giving rise to the mitochondrial DNA-encoded subunits of oxidative phosphorylation machinery. In cryo-ET, the 3D volume is reconstructed from a set of 2D projections of a frozen-hydrated specimen, which is sequentially tilted and imaged at different angles in a transmission electron microscope. In combination with subtomogram analysis, cryo-ET enables the structure determination of macromolecular complexes and their 3D organization. In this chapter, we summarize all steps required for structural characterization of mitochondrial ribosomes in situ, ranging from data acquisition to tomogram reconstruction and subtomogram analysis.