Overexpression of Tcb3-GFP in the cells was induced by addition of 1% galactose to the medium. Cells were then plunge-frozen and subjected to cryo-FIB milling. Electron cryo-tomographic tilt-series were collected on a Titan Krios (FEI) operated at 300 kV using a Quantum energy filter (slit width 20 eV) and a K2 direct electron detector (Gatan) in counting mode at a pixel size of 3.7 angstroms and at a dose rate of ~ 2-4 e-/pixel/second on the detector. The tilt-series were acquired between +60 and -56 degree starting from 0 degree with 1 degree increment using SerialEM (Mastronarde, 2005) following a grouped dose-symmetric acquisition with a group size of 4 (Bharat et al., 2018; Hagen et al., 2017), and at -4.5 micron defocus. A dose of 1.2 e-/square angstroms was applied per image of the tilt-series. The tilt-series images were reordered by tilt angle.
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