Cryo-ET of FIB-milled Cells Reveals LD Interfaces

Cryo-ET data of FIB-milled cells was acquired on three different Titan Krios microscopes (Thermo Scientific), all equipped with a K2 direct electron detector and a BioQuantum energy filter (Gatan). SerialEM was used for acquisition.²⁸ (link) To identify the positions of the lamellae, low-magnification montages of the central part of the grid were acquired , with the detector operated in linear mode. Then2.3, 5.1 or 5.5 nm pixel size montages of individual lamellae were taken and used for finding interfaces between LDs. Tilt series were acquired from 0° to ±60° (maximum) using a dose-symmetric acquisition scheme,³⁴ (link) 1° increment and a pixel size of 3.7, 3.5 or 3.4 Å. The detector was operated in counting mode. Images were acquired in a tilt group size of 4. The target defocus was set to −5 μm. The dose per tilt series image was adjusted to 1–1.2 e⁻/A² and the target dose rate at the detector was kept around 4 e⁻/px/s. For a subset of the tilt series, we used exposure fractionationation into 3 frames per tilt image. Alignment of exposure fractionated frames was performed in IMOD using alignframes. Tilt series were aligned in IMOD using patch tracking.²⁶ (link)^,27 (link) Final tomograms were reconstructed at 7.5, 7.1 or 6.7 Å pixel size using SIRT reconstruction with 10 iterations. For presentation in figure panels and movies, a median filter was applied to virtual slices. The cryo-EM data presented in the manuscript for Cidec-EGFP was obtained from 30 cells expressing Cidec-EGFP, plunge-frozen on at least 8 different days, and 15 tomograms containing LD interfaces were acquired on 10 of these cells. The cryo-EM data presented for Cidec was obtained from 15 cells expressing Cidec, frozen on 3 days, and 7 tomograms containing LD interfaces were acquired on 7 of these cells. The cryo-EM data presented of cells not expressing Cidec-EGFP was obtained from 5 cells frozen on 2 different days, and one representative tomogram, showing an LD but not containing LD interfaces, is included in the manuscript. Three of the tomograms of cells expressing Cidec-EGFP were previously used in unrelated projects published before.¹⁶ (link)^,31 (link)