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Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H₂R) in an active conformation with bound histamine and in complex with G_sheterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion into preformed nanodisc membranes using cell-free synthesis inE. colilysates. It is the first structure obtained by this detergent-free strategy and the first GPCR/G_scomplex structure in lipid environment. Structural comparison with the inactive conformation of H₂R and the inactive and G_q-coupled active state of H₁R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H₂R, its agonist-dependent internalization and its interaction with endogenously synthesized H₁R and H₂R in HEK293 cells by applying a recently developed nanotransfer technique.