Complex purification

Cell pellets were thawed in suspension buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, and 5 mM CaCl₂ supplemented with Protease Inhibitor Cocktail (TargetMol)). The complex formation was initiated by the addition of 25 mU/mL apyrase (Sigma) and 10 μM ACTH (GenScript). The suspension was incubated for 1 h at room temperature (RT). Membranes were collected by centrifugation at 30,000× g for 30 min. Complexes from membranes were solubilized by 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) supplemented with 0.1% (w/v) cholesteryl hemisuccinate (CHS, Anatrace) for 2 h at 4 °C. The supernatant was then isolated by centrifugation at 65,000× g for 30 min and incubated with M1 anti-Flag affinity resin for 2 h at 4 °C. The resin was collected by centrifugation at 500× g for 10 min and loaded onto a gravity-flow column.

The resin was then washed with 30 column volumes of washing buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 100 μM TCEP, 10 μM ACTH, 2 mM CaCl₂, 0.01% (w/v) LMNG, 0.01% (w/v) GDN, and 0.004% (w/v) CHS) before bound complexes were eluted with the same buffer containing 0.1 mg/mL Flag peptide. The complexes were then concentrated using a 100-kD Amicon Ultra centrifugal filter (Millipore) and loaded onto Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with running buffer containing 20 mM HEPES, 100 mM NaCl, 100 μM TCEP, 10 μM ACTH, 2 mM CaCl₂, pH 7.4, 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN, and 0.0002% (w/v) CHS. The sample collected from size-exclusion chromatography was analyzed by SDS–PAGE. The monomeric peak fractions were collected and concentrated to 4–6 mg/mL for electron microscopy experiments.