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| Title | Plasma FIB milling for the determination of structures in situ. |
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| Journal, issue, pages | Nat Commun, Vol. 14, Issue 1, Page 629, Year 2023 |
| Publish date | Feb 6, 2023 |
 Authors | Casper Berger / Maud Dumoux / Thomas Glen / Neville B-Y Yee / John M Mitchels / Zuzana Patáková / Michele C Darrow / James H Naismith / Michael Grange /   |
| PubMed Abstract | Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion ...Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced in a prototype microscope equipped for long cryogenic run times (> 1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication. |
 External links | Nat Commun / PubMed:36746945 / PubMed Central |
| Methods | EM (subtomogram averaging) |
| Resolution | 4.9 - 21.4 Å |
| Structure data |  EMDB-15636: Human 80S ribosome structure from pFIB-lamellae  EMDB-16185: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer: 15 to 30 nm  EMDB-16186: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer: above 30 nm matched control (for 15 to 30 nm)  EMDB-16192: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer:30 to 45 nm  EMDB-16193: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer: above 45 nm matched control (for 30 to 45 nm)  EMDB-16194: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer:45 to 60 nm  EMDB-16195: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer: above 60 nm matched control (for 45 to 60 nm)  EMDB-16196: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer: 0 to 15 nm  EMDB-16199: 80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer: above 15 nm matched control (for 0 to 15 nm) |
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Homo sapiens (human)