Phosphatidic acid

All reagents were of the highest purity available: octaethylene glycol monododecyl ether (C₁₂E₈; Nikkol Group, Nikko Chemicals Co., Ltd., Tokyo, Japan); egg yolk phosphatidylcholine (EYPC), phosphatidylethanolamine (EYPE), and phosphatidic acid (EYPA) (Avanti Polar Lipids, Alabaster, AL); all reagents used in the coupled enzyme assay include NADH, ATP, PEP, lactate dehydrogenase, and pyruvate kinase (Sigma-Aldrich, Oakville, ON Canada).

The phosphatidic acid and phosphatidylglycerol lipids were purchased from Avanti Polar Lipids. Each lipid (06:0 PG and 06:0 PA, respectively) solubilized to 15 mM in 20 mM sodium acetate (pH 5.5) was mixed with 10E8/T117v2 complex to obtain a final concentration of 10 mg/ml protein and 8 mM lipid. Crystallization screening was performed with the JCSG/IAVI/Scripps high-throughput CrystalMation robot (Rigaku) at TSRI in a sitting drop vapor diffusion format. The 10E8/T117v2 /06:0 PG crystals grew from a 1:1 (v/v) protein:reservoir solution drop equilibrated against 50% PEG 200, 0.1 M Hepes, pH 7.0, and the 10E8/T117v2 /06:0 PA crystals from a drop equilibrated against 50% PEG 400, 0.2 M NaCl, 0.1 M CHES, pH 9.5. The crystals for each complex were cryo-protected with their respective reservoir solutions.

HPLC-grade acetonitrile (ACN), methanol (MeOH), and isopropanol (IPA) were purchased from Thermo Fisher Scientific, U.S.A. HPLC-grade methyl-tert-butyl ether (MTBE), ammonium formate, and formic acid were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Ultrapure water was obtained by a Milli-Q system (Millipore, Billerica, MA). SPLASH internal standards (330707, SPLASH™ Lipidomix Mass Spec Standards) were purchased from Avanti Polar Lipids (Alabaster, U.S.A.), including lyso-phosphatidylcholine (LPC) 18:1 (d7), 25 μg/ml; lyso-phosphatidylethanolamine (LPE) 18:1 (d7), 5 μg/ml; phosphatidylcholine (PC) 15:0-18:1 (d7), 160 μg/ml; phosphatidylethanolamine (PE) 15:0-18:1 (d7), 5 μg/ml; phosphatidylglycerol (PG) 15:0-18:1 (d7), 30 μg/ml; phosphatidylserine (PS) 15:0-18:1(d7), 5 μg/ml; phosphatidylinositol (PI) 15:0-18:1 (d7), 10 μg/ml; phosphatidic acid (PA) 15:0-18:1 (d7), 7 μg/ml; sphingomyelin (SM) d18:1-18:1 (d9), 30 μg/ml; cholesterol (d7), 100 μg/ml; ceramide (Cer) 18:1 (d7), 350 μg/ml; monoglyceride (MG) 18:1 (d7), 2 μg/ml; diglyceride (DG) 15:0-18:1 (d7), 10 μg/ml; and triglyceride (TG) 15:0-18:1 (d7)-15:0, 55 μg/ml

Liposomes were prepared with phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, cholesterol, and phosphatidic acid (Avanti Polar Lipids) dissolved in chloroform at a molar ratio of 1:1:1:1.5:0.3. Nickel-charged liposomes, employed for His-tagged receptor binding, were formed by incorporating DGS-NTA-Ni [1,2-dioleoyl-sn-glycero-3-((N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl); Avanti Polar Lipids] at a final concentration of 10% (wt/wt) (18 (link)). To visualize liposome in the flotation assay, we added rhodamine B-labeled PE (Avanti Polar Lipids) into the solution at a final concentration of 0.5% (wt/wt). The mixture was evaporated using an argon gas stream to form a lipid film and then desiccated under vacuum overnight. The lipid film was hydrated in HEPES buffer and extruded through a 0.1-µm pore size membrane (Avanti Polar Lipids) to generate homogeneous liposomal populations. At room temperature, 0.43 mg/mL of fJAM-A-His was incubated with 1 mg/mL nickel-charged liposomes in a 1:10 ratio (vol/vol) for 30 min. The formation of RDLs was assessed using a flotation assay and immunoelectron microscopy. To assess the viral binding capacity of the RDL, 1 mg/mL RDLs were mixed with 1 mg/mL purified FCV at a ratio of 20:1 (vol/vol) at room temperature for 30 min. The mixture was then analyzed using a flotation assay, TEM, and cryo-EM.

Diacylglycerol (DAG, #800515) was purchased from Sigma-Aldrich. Phosphatidylcholine (PC 34:1, #850475), phosphatidylinositol (PI 34:1, #850142), phosphatidylserine (PS 34:1, #840032), phosphatidylethanolamine (PE 34:1, #850757), phosphatidylglycerol (PG 36:1, #840503), phosphatidic acid (PA 34:1, #840857), cardiolipin (CL 72:4, #710335) were purchased from Avanti polar lipids.

STZ was purchased from Sigma Chemical Co, USA. Chloroform and methanol were purchased from Merck (Merck Pte. Ltd., China). d5-Triacylglycerol (TAG)(16:0)3, d5-TAG(14:0)3, d5-TAG(18:0)3, d5-diacylglycerol (DAG)(1,3-16:0), d5-DAG(1,3-18:1), and cholesteryl-2,2,3,4,4,6-d6 octadecanoate cholesterol-26,26,26,27,27,27-d6 were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Phosphatidylinositol (PI)-d31(16:0/18:1) was obtained from Echelon Biosciences, Inc. (Salt Lake City, UT). Phosphatidylcholine (PC)-d31(16:0/18:1), phosphatidylethanolamine (PE)-d31(16:0/18:1), phosphatidylserine (PS)-d31(16:0/18:1), phosphatidic acid (PA)-d31(16:0/18:1), PA(17:0/17:0), phosphatidylglycerol (PG)-d31(16:0/18:1), lyso-bisphosphatidic acid (LBPA)-(14:0/14:0), lyso-PC(LPC)-17:0, lyso-PE(LPE)-17:1, lyso-PS(LPS)-17:1, Cer-d18:1/17:0, glucosylceramide (GluCer)-d18:1/8:0, and galactosylceramide (GalCer)-d18:1/8:0, d31-16:0, and d8-20:4 were obtained from Avanti Polar Lipids (Alabaster, AL).