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TitleFully automated, sequential focused ion beam milling for cryo-electron tomography.
Journal, issue, pagesElife, Vol. 9, Year 2020
Publish dateMar 9, 2020
AuthorsTobias Zachs / Andreas Schertel / João Medeiros / Gregor L Weiss / Jannik Hugener / Joao Matos / Martin Pilhofer /
PubMed AbstractCryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and ...Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.
External linksElife / PubMed:32149604 / PubMed Central
MethodsEM (subtomogram averaging) / EM (tomography)
Resolution30.0 Å
Structure data

EMDB-10707:
Anabaena sp. PCC 7120 Septal Junction Average from Manually Milled Lamellae
Method: EM (subtomogram averaging) / Resolution: 30.0 Å

EMDB-10708:
Anabaena sp. PCC 7120 Septal Junction Average from Lamellae generated by Automated Milling
Method: EM (subtomogram averaging) / Resolution: 30.0 Å

EMDB-10710:
Yeast Tomogram Collected on Lamella Generated by Fully Automated FIB Milling
Method: EM (tomography)

Source
  • Nostoc sp. PCC 7120 (bacteria)
  • Saccharomyces cerevisiae (brewer's yeast)

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