Raw data of the manuscript: Ligand coupling mechanism of the human serotonin transporter differentiates substrates from inhibitors Abstract: The presynaptic serotonin transporter (SERT) reuptakes the serotonin (5HT) released into the synaptic cleft, thus ensuring temporal and spatial regulation of serotonergic signalling. Clinically approved drugs used for the treatment of neurological disorders, including depression and anxiety modulate SERT by trapping the transporter in the outward-open conformation. Illicit drugs of abuse as amphetamines act as substrates but reverse the transport direction, thereby releasing intracellular accumulated 5HT. Both mechanisms increase extracellular 5HT levels. Stoichiometry of the transport cycle has been described by kinetic schemes, the structures of the main conformations within the transport cycle revealed static coordinates. By combining in-silico approaches with in-vitro experiments and making use of a homologous series of 5HT analogues, we decoded the essential coupling mechanism between the substrate and the transporter which triggers uptake. The free energy calculations showed that only scaffold-bound substrates can correctly close the extracellular gate by pulling on the bundle domain through long-range electrostatic interactions. The associated spatial and physico-chemical requirements define substrate and inhibitor properties, opening new possibilities for rational drug design approaches.