Subtomogram Averaging of Membrane Complexes in Organelle-Derived Vesicles

Cryo-electron tomography has emerged in recent years as the premier method for investigating molecular structure in situ and ex vivo, enabling structure-function relationships in a native context. In this chapter, we focus on the comprehensive investigation of membrane-associated ribosomal complexes in the context of ER-derived vesicles via cryogenic electron tomography (ET). Compared to the analysis of isolated particles, the native membrane context is conserved in this preparation, while allowing for higher sample and data throughput than in situ imaging of unperturbed cells and being amenable to biochemical manipulation. The provided protocols encompass sample preparation, cryo-ET data collection, and computational subtomogram analysis.