A large volume of literature is available on the different classes of MDR proteins found in prokaryotic and eukaryotic cells, with varying degrees of understanding of their structure and biochemistry1,2,6,7. A commonly used approach for understanding transport mechanisms is to study the effects of single mutations on function. Previous analyses have focused on either site-directed or random mutagenesis of the residues, followed by a selection screen to isolate loss- or gain-of-function mutants8,9. Both approaches, although extremely useful, have notable limitations. Site-directed mutagenesis analyzes one residue or variant at a time and is incredibly labor intensive, especially for drug transport studies, and is limited by our ability to predict crucial residues. By contrast, random mutagenesis is precisely that — random — and may also end up missing important residues. Because of these limitations, an in-depth structure–function analysis of most MDR proteins has been hampered and a consensus on how these proteins bind and export a large variety of molecules has been hard to reach.