Figure 3
Membrane binding orientation of C1. (A) Orientation of C1 on carbon films. The upper panel presents the five top 2D class averages of WT C1 on carbon. The lower panel presents the Euler angle distribution of all the particles generated by SPIDER. The sizes of the dark dots correlate with the number of particles in a certain orientation. A classical surface view coupled with the projection for the preferred orientation is shown on the right. The cartoon model indicates the general appearance of the particles on carbon film in their preferred orientation. (B) Orientation of C1 on lipid monolayers. The upper panel presents the five top 2D class averages of WT C1 bound to lipid monolayers. The lower panel presents the Euler angle distribution of all the particles generated by SPIDER. The sizes of the dark dots correlate with the number of particles in a certain orientation. Two classical surface views corresponding to the projections of the preferred orientations are shown on the left and right. The cartoon models indicate the general appearance of WT C1 particles on lipid monolayers in their preferred orientations. (C) Orientation of truncated C1 with VPS34 CTD deletion. The descriptions of the figure panels are the same as in A. (D) Flotation assay to examine the binding of C1 WT, C1 VPS34 ΔCTD and C1 ATG14L ΔBATs to liposomes containing 6% PI in a sucrose gradient (from top to bottom: 0%, 20%, 25%, 30%). All 14 fractions from top to bottom were immunoblotted using an antibody against ATG14L. (E) The flotation ratio from D for ATG14L and Supplementary information, Figure S4E for P150, VPS34 and Beclin1 was calculated by dividing the total intensity of the bands in all the fractions with the intensity of the bands in the top 8 fractions, then normalizing to the value for C1 WT. Data are represented as mean ± SD (n = 3). (F) Membrane binding model of C1. The structural areas responsible for membrane binding (ATG14L BATs domain) and the orientation of the complex (VPS34 CTD) are colored in hot pink.
