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Title | Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging. |
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Journal, issue, pages | Sci Adv, Vol. 10, Issue 17, Page eadk6285, Year 2024 |
Publish date | Apr 26, 2024 |
Authors | Maarten W Tuijtel / Sergio Cruz-León / Jan Philipp Kreysing / Sonja Welsch / Gerhard Hummer / Martin Beck / Beata Turoňová / |
PubMed 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 ...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. |
External links | Sci Adv / PubMed:38669330 / PubMed Central |
Methods | EM (subtomogram averaging) |
Resolution | 3.9 - 13.7 Å |
Structure data | EMDB-19136: Thinner is not always better: Optimising cryo lamellae for subtomogram averaging EMDB-19160: Thinner is not always better: Optimising cryo lamellae for subtomogram averaging - Lamella thickness analysis EMDB-19161: Thinner is not always better: Optimising cryo lamellae for subtomogram averaging - Ion-damage layer analysis |
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