Sodium malonate

Crystallization trials for the Ric-8A:ΔN31Gαi1:Nb8109:Nb8117:Nb8119 complex were conducted by vapor diffusion using commercially available crystallization screening kits. Sitting drops were set on 96-2 well INTELLI-PLATEs (Art Robbins Instruments) using a Gryphon crystallization robot (Art Robbins Instruments) at 3–10 mg/mL protein complex at a 1:1 v/v ratio with precipitation solution. Initial crystallization conditions were identified from hits on the ShotGun screen (MD1-88 Molecular Dimensions). Further optimization was carried out by grid screening variations in sodium malonate (Hampton Research) concentration and pH and by crystal seeding by hanging drop on 24-well VDXm plates (Hampton Research). Crystal seed stocks were prepared with the Seed Bead kit (Hampton Research) in 1.4 M sodium malonate pH 6.9. 0.9 μl of protein stock was added to 0.6 μl of reservoir and 0.3 μl of crystal seed stock and incubated at 12 °C for a minimum of 1–2 weeks. Optimal crystals were obtained from hanging drops containing 3.6 mg/ml Ric-8A:ΔN31Gαi1:3Nb, 1.4 M sodium malonate pH 6.9 at 12 °C.

N-Dodecanoyl-NBD-ceramide trihexoside and N-dodecanoyl-NBD-D-erythro-dihydrosphingosine lipid analogs were purchased from Matreya (Pleasant Gap, PA, USA). 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (NG310) detergent was purchased from Anatrace (Maumee, OH, USA). Egg white lysozyme (BP535–1), BS³ (bis(sulfosuccinimidyl)suberate crosslinker (A39266), IANBD amide (N,N’-dimethyl-N-(iodoacetyl)-N’-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine) (D2004), and the Amplex Red galactose assay (A22179) were purchased from ThermoFisher Scientific (Waltham, MA, USA). The synthetic α-galactosidase substrate p-nitrophenyl-α-D-galactopyranoside (N0877) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Human acid α-glucosidase was a gift from Genzyme (Cambridge, MA, USA). Sodium malonate was purchased from Hampton Research (Aliso Viejo, CA, USA).