Amicon centrifugation tube

The Pythium insidiosum Pi-S strain was subcultured on Sabouraud dextrose (SD) agar for 1 week. Several small pieces of agar-attaching hyphae were transferred to a 250-ml flask containing 100 ml of SD broth for shaking incubation (∼100 rpm) at 37°C for 7 days. The hyphal material was removed by filtration through a 0.2-μm-pore-size membrane (Merck Millipore, USA). The harvested hyphae were extracted to obtain SABH, as previously described (24 (link), 54 (link)). The cell-free culture broth was concentrated using an Amicon centrifugation tube (10,000 molecular weight cutoff [MWCO]; Merck Millipore) to obtain CFA, according to the reported protocol (24 (link), 54 (link)). Both SABH and CFA were stored at –30°C until use.

For reconstitution, 950 µl of lipids [4 mM POPC, 3.5 mM POPE, 2.5 mM cholesterol in 0.5%(w/v) DDM] and 1.97 ml MSP2N2 (4 mg ml⁻¹) were added to the eluted mitochondrial protein, resulting in a final molar protein:MSP2N2:lipids ratio of 1:3:150. 7.5 ml of the mixture was incubated for 1 h at room temperature before the detergent was removed by incubation with 500 mg BioBeads SM-2 (Bio-Rad) for 2.5 h. The solution was dialyzed against 20 mM HEPES pH 7.2, 20 mM KCl, 5 mM β-mercaptoethanol overnight. The sample was centrifuged for 5 min to remove aggregated protein and concentrated to a final volume of 300 µl using a 100 kDa cutoff Amicon centrifugation tube (Merck). 50 µl of sample was applied onto a Superose 6 Increase 3.2/300 gel-filtration column (GE Healthcare) and yellow complex IV fractions were collected for cryo-EM sample preparation.