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

Lena Thärichen, Robert Englmeier, Friedrich Förster^*

^*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic

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.

Original language	English
Title of host publication	Methods in Molecular Biology
Subtitle of host publication	Methods and Protocols
Editors	Antoni Barrientos, Flavia Fontanesi
Place of Publication	New York
Publisher	Humana Press
Chapter	5
Pages	75-88
Number of pages	14
Edition	1
ISBN (Electronic)	978-1-0716-3173-7
ISBN (Print)	978-1-0716-3170-6
DOIs	https://doi.org/10.1007/978-1-0716-3171-3_5
Publication status	Published - 12 May 2023

Publication series

Name	Methods in Molecular Biology
Volume	2661
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Bibliographical note

Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Funding

We would like to acknowledge Stuart Howes and the whole EM Square facility at Utrecht University for technical support in electron microscopy as well as Mihajlo Vanevic for computational technical support. We would also like to thank Gijs van der Schot and Marten Chaillet for providing pyTOM scripts. This work was supported by funding from Nederlandse Organisatie voor Wetenschappelijke Onderzoek (Vici 724.016.001). The electron microscopy within this work is part of the research program National Roadmap for Large-Scale Research Infrastructure (NEMI), project number 184.034.014, which is financed by the Dutch Research Council (NWO).

Funders	Funder number
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	184.034.014, 724.016.001

Keywords

Cryoelectron tomography
Mitochondria
Ribosome
Translation

Access to Document

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

978-1-0716-3171-3_5Final published version, 451 KBLicence: Taverne

Cite this

Thärichen, L., Englmeier, R., & Förster, F. (2023). Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation. In A. Barrientos, & F. Fontanesi (Eds.), Methods in Molecular Biology: Methods and Protocols (1 ed., pp. 75-88). (Methods in Molecular Biology; Vol. 2661). Humana Press. https://doi.org/10.1007/978-1-0716-3171-3_5

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Thärichen, L, Englmeier, R & Förster, F 2023, Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation. in A Barrientos & F Fontanesi (eds), Methods in Molecular Biology: Methods and Protocols. 1 edn, Methods in Molecular Biology, vol. 2661, Humana Press, New York, pp. 75-88. https://doi.org/10.1007/978-1-0716-3171-3_5

Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation. / Thärichen, Lena; Englmeier, Robert; Förster, Friedrich.
Methods in Molecular Biology: Methods and Protocols. ed. / Antoni Barrientos; Flavia Fontanesi. 1. ed. New York: Humana Press, 2023. p. 75-88 (Methods in Molecular Biology; Vol. 2661).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic

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T1 - Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation

AU - Thärichen, Lena

AU - Englmeier, Robert

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N2 - 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.

AB - 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.

KW - Cryoelectron tomography

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KW - Ribosome

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SN - 978-1-0716-3170-6

T3 - Methods in Molecular Biology

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

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