This study investigates the temperature-dependent structural and functional changes in the TRPM4 ion channel, a temperature-sensitive, calcium-activated cation channel. Using single-particle cryo-electron microscopy at physiological temperature (37°C), the researchers identified a 'warm' conformation of TRPM4 that differs from the 'cold' conformation observed at lower temperatures. This warm conformation is driven by a temperature-dependent calcium-binding site in the intracellular domain and is essential for TRPM4 function under physiological conditions. The study reveals that ligands such as decavanadate (a positive modulator) and ATP (an inhibitor) bind to different locations on TRPM4 at physiological temperatures compared to lower temperatures, and these binding sites have functional relevance. The researchers also elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. The study highlights the importance of studying macromolecules at physiological temperatures to understand their temperature-dependent ligand recognition and modulation. It also provides a potential molecular framework for understanding how thermosensitive TRPM channels perceive temperature changes. The findings suggest that temperature plays a critical role in TRPM4 function, with the warm conformation being essential for activation under physiological conditions. The study also shows that temperature affects the binding of ligands to TRPM4, with different binding sites observed at physiological temperatures compared to lower temperatures. The results demonstrate that temperature is a key factor in the function and regulation of TRPM4, and that studying TRPM4 at physiological temperatures is essential for understanding its role in physiological processes.This study investigates the temperature-dependent structural and functional changes in the TRPM4 ion channel, a temperature-sensitive, calcium-activated cation channel. Using single-particle cryo-electron microscopy at physiological temperature (37°C), the researchers identified a 'warm' conformation of TRPM4 that differs from the 'cold' conformation observed at lower temperatures. This warm conformation is driven by a temperature-dependent calcium-binding site in the intracellular domain and is essential for TRPM4 function under physiological conditions. The study reveals that ligands such as decavanadate (a positive modulator) and ATP (an inhibitor) bind to different locations on TRPM4 at physiological temperatures compared to lower temperatures, and these binding sites have functional relevance. The researchers also elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. The study highlights the importance of studying macromolecules at physiological temperatures to understand their temperature-dependent ligand recognition and modulation. It also provides a potential molecular framework for understanding how thermosensitive TRPM channels perceive temperature changes. The findings suggest that temperature plays a critical role in TRPM4 function, with the warm conformation being essential for activation under physiological conditions. The study also shows that temperature affects the binding of ligands to TRPM4, with different binding sites observed at physiological temperatures compared to lower temperatures. The results demonstrate that temperature is a key factor in the function and regulation of TRPM4, and that studying TRPM4 at physiological temperatures is essential for understanding its role in physiological processes.