Characterization of the dynamic resting state of a pentameric ligand-gated ion channel by cryo-electron microscopy and simulations

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological development in this family relies on high-quality structural data in multiple, subtly distinct functional states. However, structural data remain limited, particularly for the unliganded or resting state. Here we report cryo-electron microscopy structures of the ligand-gated ion channel (GLIC) under resting and activating conditions (neutral and low pH). Parallel models were built either manually or using recently developed density-guided molecular simulations. The moderate resolution of resting-state reconstructions, particularly in the extracellular domain, was improved under activating conditions, enabling the visualization of residues at key subunit interfaces including loops B, C, F, and M2–M3. Combined with molecular dynamics simulations, the cryo-electron microscopy structures at different pH describe a heterogeneous population of closed channels, with activating conditions condensing the closed-channel energy landscape on a pathway towards gating.