Extended Data Fig. 1: Biochemical and cryo-EM analyses of GGCX and the GGCX–BGP complex.
From: Structure and mechanism of vitamin-K-dependent γ-glutamyl carboxylase
a, Representative gel-filtration chromatograms of GGCX purified without any compound (blue), with menaquinone-4 (MK-4) (orange), with Anisindione (teal) and with vitamin K1 epoxide (VKO1) and Warfarin (magenta). The SDS-PAGE gels of the fractions used for cryo-EM study are shown. b, Preliminary cryo-EM maps of GGCX purified without any compound, with MK-4, and with Anisindione. The cleft of the detergent micelle in each map is indicated by a dashed line. c, Summary of cryo-EM data processing procedures of GGCX purified with VKO1 and Warfarin. d, Fourier shell correlation (FSC) curves between two half maps of GGCX purified with VKO1 and Warfarin. e, Local resolution of the cryo-EM map of GGCX purified with VKO1 and Warfarin. Maps are colored according to local resolution, estimated using cryoSPARC. f, Cryo-EM maps of the major structural elements of GGCX purified with VKO1 and Warfarin, as well as the cryo-EM map of cholesterol (CLR). TM, transmembrane helix. g, GGCX structure docked into the low-resolution cryo-EM map of GGCX purified with MK-4. The cholesterol density and the surrounding residues are shown in the lower panel. h, Comparison of the gel-filtration chromatograms of GGCX–BGP complex (green) and GGCX (blue) purified without any compound. The ratio of protein used for cryo-EM study in total protein is around 3.7% in GGCX and 23.4% in GGCX–BGP samples. The SDS-PAGE gel of the fractions of the GGCX–BGP complex used for cryo-EM study is shown. i, The SDS-PAGE gel and western blot result of the GGCX–BGP complexes expressed and purified with either MK-4 (GGCX–BGP–MK-4) or Anisindione (GGCX–BGP–ANI). The BGP bands in the SDS-PAGE gel were subjected for Mass spectrometry (MS) analysis. The western blot was performed with the same samples using the antibody specifically recognizing the Gla68 of BGP. BGPGla, carboxylated BGP protein. j, Mass spectrometry results for detecting the carboxylation of BGP in GGCX–BGP–MK-4 and GGCX–BGP–ANI samples.
