Mitochondria have an outer and inner membrane. The latter comprises an inner boundary domain and the cristae membrane, which extend into the matrix of the mitochondrion at the crista junction (CJ). The protein composition and function of both membrane and intermembrane space is central to the understanding of mitochondria. Now, Guy Perkins and colleagues (p. 3248) set out to precisely study the sub-compartmental localization of mitochondrial proteins. They use genetic tagging of the interacting proteins Mic60, Mic19 and Sam50 by adding tags that can be visualized by these factors and performing electron tomography. The authors show that Mic19 and Mic60 localized to CJs, but also formed a network along the mitochondrial periphery and are found in cristae. Furthermore, knockdown of Mic19 led to a reduction in cytochrome c oxidase subunit IV (CoxIV), and Mic19 associated with CoxIV upon Mic19 myristoylation. The localization of Mic19 and Mic60 incompletely overlapped at CJs with Sam50, which is not uniformly distributed around the mitochondrial periphery. Taken together, this analysis provides evidence at nanoscale resolution for the separate localization of mitochondrial partner proteins. It clarifies the position of key factors of the mitochondrial intermembrane space, the disruption of which is implicated in human neurodegenerative diseases.