Cryo-EM structure of cell-free synthesized human histamine 2 receptor/G_s complex in nanodisc environment

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_s heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. 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.