Significance EmrE is a small membrane transporter found in Escherichia coli that exports drug-like molecules from the cell, contributing to antibiotic resistance. In EmrE, as well as in the wider small-multidrug resistance transporter family, a specific anionic amino acid (E14) has been implicated in governing the conformational changes that export drugs. However, due to sparse structural information, the exact interactions remain unidentified. Through interactive molecular dynamics to incorporate existing cryo-electron microscopy data, we create a fully refined atomic model of EmrE. We then embed this model in a lipid bilayer and evaluate the interactions within EmrE under different loading states. We find that E14 makes specific hydrogen bonds to neighboring residues, coupling observed experimental phenomena to interactions at the atomic scale.