Nanodisc Formation

Freshly purified 3a dimer in Buffer 1 was reconstituted into MSP1E3D1 nanodiscs with a mixture of lipids (DOPE:POPS:POPC at a 2:1:1 mass ratio, Avanti, Alabaster, Alabama) at a final molar ratio of 1:4:400 (Monomer Ratio: 3a, MSP1E3D1, lipid mixture). First, 20 mM solubilized lipid in Nanodisc Formation Buffer (20 mM HEPES, 150 mM KCl, 1 mM EDTA pH 7.4) was mixed with additional DDM detergent and 3a protein. This solution was mixed at 4°C for 30 minutes before addition of purified MSP1E3D1. This addition brought the final concentrations to approximately 15 μM 3a, 60 μM MSP1E3D1, 6 mM lipid mix, and 10 mM DDM in Nanodisc Formation Buffer. The solution with MSP1E3D1 was mixed at 4°C for 10 minutes before addition of 200 mg of Biobeads SM2. Biobeads (washed into methanol, water, and then Nanodisc Formation Buffer) were weighed after liquid was removed by pipetting (damp weight). This mix was incubated at 4°C for 30 minutes before addition of another 200 mg of Biobeads (for a total 400 mg of Biobeads per 0.5 mL reaction). This final mixture was then gently tumbled at 4°C overnight (~ 12 hours). Supernatant was cleared of beads by letting large beads settle and carefully removing liquid with a pipette. Sample was spun for 10 minutes at 21,000 × g before loading onto a Superdex 200 increase column in 20 mM HEPES, 150 mM KCl, pH 7.4. Peak fractions corresponding to 3a protein in MSP1E3D1 were collected, 10 kDa cutoff spin concentrated and used for grid preparation. MSP1E3D1 was prepared as described⁵⁵ without cleavage of the His-tag. Tetrameric 3a in nanodiscs was prepared similarly, except with a ratio of 1:2:200 (Monomer Ratio: 3a, MSP1E3D1, lipid mixture).