In vitro characterization of SHIP164. (A) (a) Following batch purification using anti-FLAG M2 resin, 3XFLAG-SHIP164_Δ901–1099 was further purified by size exclusion chromatography on a Superdex-200 column. The gel filtration profile is shown (black), along with that for molecular weight standards (light blue). (b) Sample purity was examined by SDS-PAGE. (c) Representative negative stain electron micrograph of 3XFLAG-SHIP164_Δ901–1099 (50 nM) using FEI Tecnai 12 microscope at 120 kV at a nominal magnification of 52,000×. Staining was with 2% uranyl acetate on carbon-coated copper grids that were glow-discharged for 30 s at 22 mA. Green circles represent particles manually picked with a 300 Å mask diameter in RELION-3.1. (B) Cryo-EM workflow. Micrographs, including the representative one shown, were collected using Titan Krios G2 transmission electron microscope (Thermo Fisher Scientific) at 300 kV equipped with a K3 summit direct detection camera (Gatan). Green circles represent particles manually picked for initial reference-free 2D classification prior to autopicking. (C) The processing workflow yielded a reconstruction with an estimated resolution of 8.3 Å according to the Fourier shell correlation = 0.143 criterion. Source data are available for this figure: SourceData FS1.