Cryo-electron tomography for the structural study of mitochondrial translation

摘要

Cryo-electron tomography (cryo-ET) enables the three-dimensional (3D) structural characterization of macromolecular complexes in their physiological environment. Thus, cryo-ET is uniquely suited to study the structural basis of biomolecular processes that are extremely difficult or even impossible to reconstitute using purified components. Translation of mitochondrial genes, which occurs in the secluded interior of mitochondria, falls into this category. Here, we describe the principles of cryo-ET in the context of mitochondrial translation and outline recent developments and challenges of the method. The 3D image of a frozen-hydrated biological sample is computed from its 2D projections, which are acquired using a transmission electron microscope. In conjunction with automated detection of different copies of the molecule of interest and averaging of the corresponding subtomograms, cryo-ET enables macromolecular structure determination in the native environment (i.e. in situ) at sub-nanometer resolution. The preservation of the native environment furthermore allows the extraction of contextual information about the molecules, including the location of specific molecules with respect to membranes, their relative positioning and the spatial organization with respect to other types of macromolecules. Recent preparative developments extend the field of application of cryo-ET from isolated organelles to cultured eukaryotic cells and even tissue, making the traditional borders between molecular and cellular structural biology disappear.