Single-Particle Cryo-EM sample vitrification

Reconstituted complexes were concentrated to 7-10 μM by Amicon Ultra −4 centrifugal filter (Ultracel 50K, Millipore). Fresh detergent (CHAPS, 0.25%-0.5%) was added before grid preparation. In order to minimize sample interaction with the air-water interface and to keep the complex intact²⁹, the complex was vitrified using a commercial prototype (from TTP Labtech) of the Spotiton robot^14,30,31. The time between sample being spotted onto the nanowire grid³² and vitrification into liquid ethane was 133 ms. Grids were plasma-cleaned (Gatan Solarus) for 5 s using an H2/O2 mixture prior to vitrification. The robot chamber was operated at room temperature and ~80% humidity in the sample and plunging area.