Purification of the Active-State G Protein-Coupled Receptor ADGRL4 for Cryo-Electron Microscopy Using a Modular Tag...

ADGRL4 is an adhesion G protein-coupled receptor (aGPCR) implicated in tumour progression in multiple malignancies. We recently determined the first cryo-EM structure of active-state ADGRL4, revealing its weak coupling to the heterotrimeric G protein G_q and providing insights into its activation mechanism. Here, we describe a complete modular workflow for purifying active-state ADGRL4 over 2-3 days using a multifunctional tagging strategy incorporating multiple orthogonal detection, purification, and cleavage tags at the N-terminus as well as a tethered mini-G_q at the C-terminus. This configuration enhanced receptor cell-surface expression and stability and allowed different purification strategies to be tested during the development of the purification protocol. Although developed and optimised for ADGRL4, this approach is readily transferable to other weakly coupling aGPCRs or GPCRs where complex stability is a limiting factor for structural analysis. Key features • Complete workflow for purifying active-state ADGRL4 for cryo-EM analysis. • Modular, multifunctional N-terminal tagging strategy supporting multiple orthogonal purification and detection methods without any negative effect on cell surface expression levels. • Tethered mini-G_q increases stability and receptor cell-surface expression. • Modular purification tagging configuration provides freedom to change purification methodologies without having to perform additional receptor engineering or cloning.