Cryo-EM Grid Preparation for Protein and Cells

For cryo-EM grid preparation of purified protein, 2.5 µL of the specimen was applied to a freshly glow-discharged Quantifoil R2/2 Cu/Rh 200 mesh grid, adsorbed for 60 s, blotted for 4 to 5 s, and plunge-frozen into liquid ethane in a Vitrobot Mark IV (ThermoFisher), while the blotting chamber was maintained at 100% humidity at 10 °C. Grids for cryo-FIB milling of D. radiodurans cells were prepared as described previously (10 (link)). Briefly, D. radiodurans strain BAA-816 (obtained from the American Type Culture Collection) was grown aerobically in TGY liquid medium (49 (link)). Cells were grown for 24 h at 30°C prior to harvesting and staining with FM4-64 fluorescent membrane dye (Invitrogen). Four microliter of cells was loaded on Finder grids (Electron Microscopy Sciences) and plunge-frozen in a liquid ethane–propane mixture kept at liquid nitrogen temperatures using a Vitrobot Mark IV (Thermo Fisher Scientific). Grids were clipped and stored under liquid nitrogen.