Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), is one of the deadliest human pathogen. Despite existing chemotherapy, Mtb has been responsible for the death of 1.4 million people and about 10 million new infections in 2015 (WHO report on TB, 2016). This concerns not only developing countries since 5000 new cases are reported yearly in France. The current problems in TB eradication are: lengthy treatments, co-infection with HIV and emergence of multidrug-resistant strains of Mtb. During the last 50 years, very few antitubercular drugs have been discovered and put on the market. Therefore, identifying new molecules targeting mycobacteria is urgent although highly challenging. Most mycobacteria are naturally resistant to antibiotics for which the highly hydrophobic cell wall represents an impermeable barrier. Mycolic acids (MA) are very long lipids made of 90 carbon atoms that are essential components of the mycomembrane and contribute to the high hydrophobicity of the cell wall. MA are synthesized in the cytoplasm and then transported to the periplasm by a specific transporter, MmpL3, which belongs to the superfamily of Resistance-Nodulation-Division permeases. To date, our knowledge about the mechanism by which MA are transported by MmpL3 remains very limited, due to the lack of both in vitro characterization and structural information. The fact that MmpL3 is essential for mycobacterial growth makes it an extremely attractive drug target for future translational applications. Recent whole-cell-based screening conducted by several independent teams, including ours, led to the identification of various chemical entities exhibiting potent antitubercular activity. The mode of action of all these chemotypes involves the inhibition of MA transport to the bacterial surface. In most studies, MmpL3 was designated as the primary target based on the presence of mutations occurring in mmpL3 in spontaneous resistant strains. Among them, some have already reached phases II or III of clinical trials and/or have shown to exhibit synergetic effects with existing antitubercular drugs. Despite these exciting promises, concerns have recently been raised regarding the real implication of MmpL3 as the target of many of these compounds as well as their mechanism of action. Therefore, describing, at a molecular and structural level, the MmpL3-mediated transport mechanism might help to understand how MA are translocated to the cell surface and to validate the importance of MmpL3 in cell wall assembly. This may also greatly help to describe the mode of action of some of the recently identified MmpL3 inhibitors. The objectives of MyTraM consists of the 1) expression and purification of large amounts of recombinant MmpL3; 2) implementation of hybrid structural biology approaches including X-ray crystallography and cryo-electron microscopy to determine the three-dimensional structure(s) of MmpL3; 3) development of an innovative biochemical in vitro assay to assess MA transport and to investigate the mode of action of several MmpL3 inhibitors; and 4) determination of the binding constants of MmpL3 substrates and inhibitors in solution using microscale thermophoresis. We anticipate that this 3-year project should add important breakthroughs in our understanding of MmpL structure-function relationships and lead to a more precise description of the mode of inhibition of a family of promising anti-TB compounds. On a longer term, these studies should also aid in the future improvement of already existing MmpL3 inhibitors and in the conception of new generations of MmpL3-based drugs for the treatment of TB and other mycobacterial infections.