Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation

Lena Thärichen; Robert Englmeier; Friedrich Förster

doi:10.1007/978-1-0716-3171-3_5

Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation

Methods Mol Biol. 2023:2661:75-88. doi: 10.1007/978-1-0716-3171-3_5.

Authors

Lena Thärichen¹, Robert Englmeier¹, Friedrich Förster²

Affiliations

¹ Structural Biochemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
² Structural Biochemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, Utrecht, The Netherlands. f.g.forster@uu.nl.

PMID: 37166632
DOI: 10.1007/978-1-0716-3171-3_5

Abstract

Cryoelectron tomography is a method to image biological samples three-dimensionally at molecular resolution. This modality provides insights into intracellular processes in their physiological settings. Obtaining a high-quality sample for cryoelectron tomography on mitochondria, however, can be challenging. In this chapter, we describe the crucial steps from sample preparation to data acquisition enabling studies of mitochondrial translation in situ by cryoelectron tomography. We provide detailed protocols for yeast and human mitochondria preparations yielding a high concentration of intact mitochondrial vesicles on cryo-EM grids. In addition, we describe a workflow for particle identification and spatial mapping in context of the organelle.

Keywords: Cryoelectron tomography; Mitochondria; Ribosome; Translation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cryoelectron Microscopy / methods
Electron Microscope Tomography / methods
Humans
Mitochondria* / metabolism
Protein Biosynthesis*
Saccharomyces cerevisiae
Specimen Handling / methods