(A) Domain structures of the four subunits of PI3KC3-C1. The small unlabeled yellow box between VPS34 C2 and helical domains is the CHIL motif, described below. (B) Purification of PI3KC3-C1. Coomassie-stained SDS-PAGE gel of purified PI3KC3-C1. (C) Thin-layer chromatography of radiolabeled PI(3)P generated by PI3KC3-C1 from PI and [γ-32 P] ATP. (D) Reference-free class averages of PI3KC3-C1, each containing ∼200 particles. The first row represents particles that were selected for the 3D reconstruction. The lower two rows represent particles excluded from the 3D reconstruction, the arrowhead indicating the position of a density dislodged from the main part of the complex. All class averages shown are calculated from data acquired on untilted grids, except the top right class average which is from 45° tilt. (E) 3D reconstruction calculated from ∼39,000 particles in four orientations and displayed at a threshold determined from subsequent docking analyses.