Subtomogram averaging by cryo electron tomography using CRYO ARM™ 300 II for purified and cellular samples

Fumiaki Makino; Haruaki Yanagisawa; Fabian Eisenstein; Tomoko Miyata; Miki Kinoshita; Masahide Kikkawa; Keiichi Namba; Fumiaki Makino; Haruaki Yanagisawa; Fabian Eisenstein; Tomoko Miyata; Miki Kinoshita; Masahide Kikkawa; Keiichi Namba

doi:10.3934/biophy.2026008

AIMS Biophysics

2026, Volume 13, Issue 2: 131-142. doi: 10.3934/biophy.2026008

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Subtomogram averaging by cryo electron tomography using CRYO ARM™ 300 II for purified and cellular samples

1.
JEOL Ltd., Akishima, Tokyo 196-8558, Japan
2.
Graduate School of Frontier Biosciences, The University of Osaka, Suita, Osaka 565-0871, Japan
3.
JEOL YOKOGUSHI Research Alliance Laboratories, The University of Osaka, Suita, Osaka 565-0871, Japan
4.
Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
5.
Present address: DECTRIS AG, 5405 Baden-Daettwil, Switzerland

Received: 21 November 2025 Revised: 11 March 2026 Accepted: 20 March 2026 Published: 27 March 2026

Cryo electron tomography (cryo-ET) enables three-dimensional visualization of biological macromolecules in their near-native environments, bridging the gap between structural and cellular biology. In this study, we evaluated high-resolution subtomogram averaging (STA) using a cryo-TEM (CRYO ARM™ 300 II, JEOL) and PACEtomo software system for both single-particle analysis (SPA) type grids and lamellae prepared by cryo-focused ion beam (cryo-FIB) milling (JIB-4700F, JEOL). Using mouse apoferritin and E. coli ribosomes in thin vitreous ice film, resolutions of 2.04 Å and 2.86 Å were achieved, respectively. Using cryo-FIB lamellae of Salmonella cells, in situ ribosome structures were resolved to 8.2 Å resolution.
- cryo-EM,
- cryo-ET,
- STA,
- cryo-FIB-SEM,
- CryoLameller,
- CRYO ARM,
- structural cell biology
Citation: Fumiaki Makino, Haruaki Yanagisawa, Fabian Eisenstein, Tomoko Miyata, Miki Kinoshita, Masahide Kikkawa, Keiichi Namba. Subtomogram averaging by cryo electron tomography using CRYO ARM™ 300 II for purified and cellular samples[J]. AIMS Biophysics, 2026, 13(2): 131-142. doi: 10.3934/biophy.2026008

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Abstract

Cryo electron tomography (cryo-ET) enables three-dimensional visualization of biological macromolecules in their near-native environments, bridging the gap between structural and cellular biology. In this study, we evaluated high-resolution subtomogram averaging (STA) using a cryo-TEM (CRYO ARM™ 300 II, JEOL) and PACEtomo software system for both single-particle analysis (SPA) type grids and lamellae prepared by cryo-focused ion beam (cryo-FIB) milling (JIB-4700F, JEOL). Using mouse apoferritin and E. coli ribosomes in thin vitreous ice film, resolutions of 2.04 Å and 2.86 Å were achieved, respectively. Using cryo-FIB lamellae of Salmonella cells, in situ ribosome structures were resolved to 8.2 Å resolution.

Acknowledgments

We thank Radostin Danev for his valuable advice and assistance in setting up and optimizing the PACEtomo system in our CRYO ARM™ 300 II, Yoshie Kushima for help in establishing lamella preparation workflow by cryo-FIB-SEM. We also thank Radostin Danev for his careful reading of the manuscript and for providing insightful and constructive suggestions. This work has been supported by Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED under Grant Numbers JP24am121003 (to K.N.), by the Cyclic Innovation for Clinical Empowerment from AMED under Grant Number JP17pc0101020 (to K.N.), and by JEOL YOKOGUSHI Research Alliance Laboratories of the University of Osaka (to K.N.).

Conflict of interest

The products (CRYO ARM™) described and utilized in this manuscript are proprietary products of JEOL, the employer of (some of) the authors.

Author contributions

F.M. and K.N. conceptualized, administered, and supervised the project. F.M., and H.Y. and F.E. designed the experiments. T.M, M.K. and H.Y. prepared protein and cell samples. T.M., M.K., F.E., F.M., and H.Y. performed experiments with cryo-FIB-SEM and data collection with a cryo-TEM. H.Y. conducted the STA image analysis. F.M. wrote the initial draft of the manuscript. All authors reviewed, edited, and approved the final version of the manuscript.

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