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TitleThinner is not always better: Optimizing cryo-lamellae for subtomogram averaging.
Journal, issue, pagesSci Adv, Vol. 10, Issue 17, Page eadk6285, Year 2024
Publish dateApr 26, 2024
AuthorsMaarten W Tuijtel / Sergio Cruz-León / Jan Philipp Kreysing / Sonja Welsch / Gerhard Hummer / Martin Beck / Beata Turoňová /
PubMed AbstractCryo-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 linksSci Adv / PubMed:38669330 / PubMed Central
MethodsEM (subtomogram averaging)
Resolution3.9 - 13.7 Å
Structure data

EMDB-19136: Thinner is not always better: Optimising cryo lamellae for subtomogram averaging
Method: EM (subtomogram averaging) / Resolution: 3.9 Å

EMDB-19160: Thinner is not always better: Optimising cryo lamellae for subtomogram averaging - Lamella thickness analysis
Method: EM (subtomogram averaging) / Resolution: 4.5 Å

EMDB-19161: Thinner is not always better: Optimising cryo lamellae for subtomogram averaging - Ion-damage layer analysis
Method: EM (subtomogram averaging) / Resolution: 13.7 Å

Source
  • Dictyostelium discoideum AX2 (eukaryote)

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