R1.2 1 3 400 mesh au holey carbon grids

PORCN samples (~8 mg/ml) with 10 μM LGK974 and HHAT–SHH-N–Fab^3H02 complex (~10mg/ml) were applied to Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil), respectively. For the preparation of palmitoleoyl-CoA-bound PORCN samples, palmitoleoyl-CoA was added into the apo-PORCN sample at the final concentration of 1 mM before vitrification. For the preparation of LGK974/WNT3Ap-bound PORCN samples, WNT3Ap (MHLKCKCHGLSGSCEVKTCWWS, C5-C19, C7-C14, Biomatik) were mixed with LGK974-bound PORCN at a final concentration of 1 mM. For the preparation of product-bound PORCN, pamWNT3Ap was added into the apo-PORCN at the final concentration of 1.2 mM. The above two peptide mixtures were incubated on ice for 30 min before vitrification. The grids were blotted and plunged into liquid ethane for flash freezing using a Vitrobot Mark IV (FEI). The grids were imaged in a 300 kV Titan Krios (FEI) with a Gatan K3 Summit direct electron detector (Gatan). Data were collected using SerialEM ^{45 (link)} at 0.83 Å/pixel or 0.842 Å/pixel. Images were recorded for 5-second exposures in 50 subframes with a total dose of ~60 electrons per Ų.

The ~6 mg/ml EBI2–G_i–scFv16 complex and ~12 mg/ml EBI2-BRIL–Fab–Nb complex were used for cryo-EM studies, respectively. 3 μl of freshly purified protein complex was applied to Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil). Excess sample was blotted with filter paper for 4 seconds before plunge-freezing in liquid ethane using a Vitrobot Mark IV (FEI) at 20 °C and 100% humidity. The grids were imaged on a 300 keV Titan Krios (FEI) with a Gatan K3 Summit direct electron detector (Gatan). Data were collected in super-resolution mode with SerialEM (Mastronarde, 2005 (link)) and the collection parameters of each dataset were summarized in the Table S1.