This study investigates how ligand efficacy modulates the conformational dynamics of the μ-opioid receptor (μOR), a key target for pain management. Using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), the researchers show how ligand-specific conformational changes in μOR translate into varying intrinsic efficacies at the transducer level. They identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation capable of G-protein binding and a fully activated conformation that reduces GDP affinity in the ternary complex. The interaction of β-arrestin-1 with the μOR core binding site is less specific and occurs with lower affinity than G-protein binding. μOR is a G-protein-coupled receptor (GPCR) and an important drug target for analgesia. However, activation by opioids like morphine and fentanyl can lead to adverse effects, including constipation, tolerance, and respiratory depression. μOR activates G-protein family G proteins and recruits β-arrestins. Previous studies suggested that μOR's analgesic effects were mediated by G-protein signaling, while respiratory depression was mediated by β-arrestin recruitment. G-protein-biased agonists were expected to have reduced side effects, but recent studies show that overly strong G-protein signaling (super-efficacy) is responsible for respiratory depression, while partial agonists with lower efficacy provide a safer profile. High-resolution structures provide insights into μOR activation and G-protein signaling. The C-terminal helix of G binds to an opening in the cytoplasmic surface of the 7-transmembrane helix bundle, formed by an approximately 10-Å outward movement of the intracellular end of transmembrane helix 6. Structures of μOR in complex with β-arrestin are not yet available due to the lack of a stable or structurally homogeneous complex. However, structures determined by X-ray crystallography and cryo-electron microscopy generally represent snapshots of the most stable and homogeneous conformations. The study combines DEER and smFRET to investigate the molecular basis of μOR activation and signal transfer. They examined the effect of nine representative μOR ligands on the conformation and dynamics of TM6, including antagonists, G-protein-biased agonists, and super-efficacy agonists. The results show how the conformational ensemble of μOR is fine-tuned by ligand binding, resulting in distinct efficacies and signal bias. The study reveals that the conformational heterogeneity of μOR is modulated by ligand binding, with different ligands stabilizing different conformations. The results suggest that the conformational changes in μOR are crucial for G-protein and β-arrestin binding and signaling. The study also highlights theThis study investigates how ligand efficacy modulates the conformational dynamics of the μ-opioid receptor (μOR), a key target for pain management. Using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), the researchers show how ligand-specific conformational changes in μOR translate into varying intrinsic efficacies at the transducer level. They identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation capable of G-protein binding and a fully activated conformation that reduces GDP affinity in the ternary complex. The interaction of β-arrestin-1 with the μOR core binding site is less specific and occurs with lower affinity than G-protein binding. μOR is a G-protein-coupled receptor (GPCR) and an important drug target for analgesia. However, activation by opioids like morphine and fentanyl can lead to adverse effects, including constipation, tolerance, and respiratory depression. μOR activates G-protein family G proteins and recruits β-arrestins. Previous studies suggested that μOR's analgesic effects were mediated by G-protein signaling, while respiratory depression was mediated by β-arrestin recruitment. G-protein-biased agonists were expected to have reduced side effects, but recent studies show that overly strong G-protein signaling (super-efficacy) is responsible for respiratory depression, while partial agonists with lower efficacy provide a safer profile. High-resolution structures provide insights into μOR activation and G-protein signaling. The C-terminal helix of G binds to an opening in the cytoplasmic surface of the 7-transmembrane helix bundle, formed by an approximately 10-Å outward movement of the intracellular end of transmembrane helix 6. Structures of μOR in complex with β-arrestin are not yet available due to the lack of a stable or structurally homogeneous complex. However, structures determined by X-ray crystallography and cryo-electron microscopy generally represent snapshots of the most stable and homogeneous conformations. The study combines DEER and smFRET to investigate the molecular basis of μOR activation and signal transfer. They examined the effect of nine representative μOR ligands on the conformation and dynamics of TM6, including antagonists, G-protein-biased agonists, and super-efficacy agonists. The results show how the conformational ensemble of μOR is fine-tuned by ligand binding, resulting in distinct efficacies and signal bias. The study reveals that the conformational heterogeneity of μOR is modulated by ligand binding, with different ligands stabilizing different conformations. The results suggest that the conformational changes in μOR are crucial for G-protein and β-arrestin binding and signaling. The study also highlights the