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Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging

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Tuijtel,  Maarten       
Department of Molecular Sociology, Max Planck Institute of Biophysics, Max Planck Society;

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Cruz-León,  Sergio       
Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Max Planck Society;

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Kreysing,  Jan Philipp       
Department of Molecular Sociology, Max Planck Institute of Biophysics, Max Planck Society;
IMPRS-CBP, Max Planck Institute of Biophysics, Max Planck Society;

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Welsch,  Sonja       
Central Electron Microscopy Facility, Max Planck Institute of Biophysics, Max Planck Society;

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Hummer,  Gerhard       
Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Max Planck Society;
Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main, Germany;

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Beck,  Martin       
Department of Molecular Sociology, Max Planck Institute of Biophysics, Max Planck Society;
Institute of Biochemistry, Goethe University Frankfurt, Frankfurt am Main, Germany;

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Turoňová,  Beata       
Department of Molecular Sociology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Tuijtel, M., Cruz-León, S., Kreysing, J. P., Welsch, S., Hummer, G., Beck, M., et al. (2024). Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging. Science Advances, 10(17): eadk6285. doi:10.1126/sciadv.adk6285.


Cite as: https://hdl.handle.net/21.11116/0000-000F-38F6-D
Abstract
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo-focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.