Extended Data Fig. 1: Workflow of cryo-EM data processing of PI3Kγ complexed with Gβγ and ADP.
From: Molecular basis for Gβγ-mediated activation of phosphoinositide 3-kinase γ
The un-tilted (3525 frames, black scale bar = 100 nm) and 30° tilted datasets (2943 frames, scale bar = 100 nm) were collected and combined for data processing in CryoSPARC, resulting in 1103979 particles for further classification using RELION 3D classification with 3 classes. Particles in Class1 showing weaker GβγCTD density were further processed via CryoSPARC 3D classification. Most particles without the GβγCTD density classified into two species: PI3Kγ·ADP and PI3Kγ·ADP–GβγHD. The remaining particles containing the GβγCTD were combined with particles from RELION Classes 2 & 3, containing both GβγCTD and GβγHD, and subjected to masked 3D classification in CryoSPARC, resulting in two distinct conformations of PI3Kγ·ADP–GβγHD–GβγCTD, states 1 and 2, with GβγCTD and the associated p101 CTD exhibiting the largest variation.
