Numerical values in tabular format of the processed data used for plotting the respective figure panels. For orientation, see the data organization outlined below. Organization of data: • Fig. 1C: Membdensity_* Each .csv file contains the mean density of membrane voxels, normalized to mitochondrial volume, per tomogram. One file is provided for each condition. • Fig. 1D: Membdensity_distDep_* Each .csv file contains the mean density of membrane voxels, normalized to mitochondrial volume, per tomogram. One file is provided for each distance range from the aggregate. • Fig. 2B: PartAbundQuant_perCond_* Each .csv file contains the mean density of particles, normalized to mitochondrial volume, per tomogram. One file is provided for each particle species and condition. • Fig. 2E: PartAbundQuant_perCond_* Each .csv file contains the mean density of particles, normalized to mitochondrial volume, per tomogram. One file is provided for each particle species and condition. • Fig. 2H: PartEnrichment2Agg_* Each .csv file contains the mean density of particles, normalized to mitochondrial volume, per tomogram. One file is provided for each particle species and distance range from the aggregate.

Cells have evolved organelle-specific responses to maintain protein homeostasis (proteostasis). During proteostatic stress, mitochondria downregulate translation and enhance protein folding, yet the underlying mechanisms remain poorly defined. Here, we employed cryo-electron tomography to observe the structural consequences of mitochondrial proteostatic stress within human cells. We detected protein aggregates within the mitochondrial matrix, accompanied by a marked remodeling of cristae architecture. Concomitantly, the number of mitochondrial ribosome complexes was significantly reduced. Mitochondrial Hsp60 (mHsp60), a key protein folding machine, underwent major conformational changes to favor complexes with its co-chaperone mHsp10. We visualized the interactions of mHsp60 with native substrate proteins, and determined in vitro mHsp60 cryo- EM structures enabling nucleotide state assignment of the in situ structures. These data converge on a model of the mHsp60 functional cycle and its essential role in mitochondrial proteostasis. More broadly, our findings reveal structural mechanisms governing mitochondrial protein biosynthesis and their remodeling under proteostatic stress.