1 palmitoyl 2 oleoyl glycero 3 phosphocholine

1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, referred to as PC here), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG, referred to as PG here), and 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissaminerhodamine B sulfonyl) (RhB-PE) were purchased from Avanti Polar Lipids and used as received. Calcein was obtained from Sigma–Aldrich. Peptides with pre-designed amino acid sequences were ordered from SynPeptide Co., Ltd. (Nanjing, China; >95% purity). All experiments were carried out at room temperature at 22 °C.

The lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLiPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids.
Giant unilamellar vesicles (GUVs) were generated employing the gentle agarose swelling method^{20 (link),67 (link)}. Briefly, lipid mixtures containing POPS:DLiPC:DPPC:CHIM-L (14.29:42.85:28.57:14.29) were prepared from 5 mM stock solutions and added to a thin film of agarose, which was prepared in a two-well ibidi slide by addition of a 1% w/v solution of ultra-low gelling agarose in double distilled H₂O at 60 °C. The mixture was allowed to dry for 30 min in vacuum and was then equilibrated in HEPES buffer for 1 h, followed by clicking with AF488 (5 µM) and labeling with FAST DiI (5 µM).

Membrane scaffold protein (MSP) 1D1 was expressed and purified following an established protocol (Martens et al., 2016 (link)). For lipid preparation, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, Avanti Polar Lipids), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE, Avanti Polar Lipids) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG, Avanti Polar Lipids) were mixed at a molar ratio of 3:1:1, dried under Argon and resuspended with 14 mM DDM (Autzen et al., 2018 (link)). For nanodisc reconstitution, HsFpn, MSP1D1 and lipid mixture were mixed at a molar ratio of 1:2.5:50 and incubated on ice for 1 hr. Detergents were removed by the sequential addition of 60 mg/mL Biobeads SM2 (Bio-Rad) for three times over a 3-hr period. The sample was then incubated with Biobeads overnight at 4 °C. After removal of Biobeads, 11F9 was added to the sample at a molar ratio of 1.1:1 to HsFpn. The complex was incubated on ice for 30 min before being loaded onto a SEC column equilibrated with the FPLC buffer without detergent. The purified nanodisc sample was concentrated to 10 mg/ml and incubated with 2 mM CaCl₂ for 30 min on ice before cryo-EM grid preparation.