Mitochondrial Proteostasis Cryo-ET Analysis This repository contains the code used for the study: Ehses et al., Structural remodeling of the mitochondrial protein biogenesis machinery upon proteostatic stress The code was written in MATLAB 2019b and used to process and analyze cryo-electron tomography (cryo-ET) data. Below you will find an overview of the repository structure and usage. ⸻ Repository Structure 1_Processing_PROCMAN Disclaimer: This module is heavily inspired by Will Wan’s TOMOMAN (TOMOgram-MANager) (W. Wan et al., 2024). It follows a similar philosophy and reuses parts of TOMOMAN with modifications. The name PROCMAN (PROCessing-MANager) is a direct homage. Submodules typically include a _prepare script, which outputs a parameter file, and a corresponding _execute script, which performs the actual computation. Exceptions are PROCbox.m and PROCbridge.m. Submodules: • PROCMAN: Basic preprocessing steps, from raw frames or SNARtomo data to reconstructed tomograms. • PROCdyno: Dynamo template matching, execution, and particle extraction, with options for custom masking. • PROCgap: STOPGAP equivalent of PROCdyno. (Not used in the published study.) • PROCbridge: Conversion utilities between platforms and functions for data archiving. • PROCbox: Handy helper functions, e.g. for generating subsets from PROCMAN tomolists. Folder structure: • level0_appendix: Associated files (pretrained models, submission headers, etc.) • level1_functions: Core functions, called by the submodules • level2_functions: Third-party functions required for level1 ⸻ 2_Analysis_MuRePP MuRePP stands for Multidimensional Representation of Particle Properties. Like module 1, it includes _prepare and _execute scripts. • _prepare: Performs the main analysis steps and generates parameter files. • _execute: Visualizes the results, mainly through plotting. Outputs are RELION-style .star files with additional fields. These can be used for visualization in ChimeraX via ArtiaX. ⸻ 0_Individual_scripts Standalone scripts for specific tasks (e.g. RELION, WARP, Deepfinder). They do not interact with each other. A short description is provided at the beginning of each script. ⸻ Prerequisites A number of tools is required for successful run of the scripts: • tomoman • topaz • stopgap • relion • membrain • isonet • dynamo • surface morphometrics • masktomrec • aretomo • ctffind4 • snartomopace Additionally, paths and login information needs to be set to match your required data structure and computing environment.

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.. Static version of https://gitlab.gwdg.de/fernandez-busnadiego-lab/mitochondrial-proteostasis-cryo-et-analysis