R1.2 1 3 au 300 mesh

Acetone pre-washed electron microscopy grids (Ultrafoil R1.2/1.3 Au 300 mesh) were glow-discharged and 3 μL of the sample was applied to the grid in a Vitrobot Mark IV chamber (Thermo Fisher Scientific), set to 100% humidity at 4 °C. The sample was blotted for 10 s with a blot force of 19 and then flash frozen in liquid ethane.

The sample was prepared using a Quantifoil R 1.2/1.3 Au 300 mesh holey-carbon grid and plunged using a Thermo Fisher Scientific Vitrobot cryo-plunger. The grid was glow discharged at −15 mA for 1 min (PELCO easiGlow) before protein solution was applied. ScMet18 (untagged, His-SUMO tag removed by SUMO protease treatment) was diluted to a final concentration of 1.2 mg/mL (9 µM) in Buffer C (50 mM HEPES pH 7.5, 100 mM NaCl, 1% glycerol, and 1 mM BME) and 33 mM sodium acetate was added to the solution. The use of this buffer composition and this protein concentration was empirical; it allowed for good distribution of ScMet18 particles on grids, limiting the crowding of particles. ScMet18 (3 μL) was applied to the grid and was blotted for 6 sec before plunging into liquid ethane and transferring to storage buttons. The temperature and humidity were 10 °C and 95%, respectively.