MMG is among the most immunostimulatory lipids found in the cell envelope of the Mycobacterium bovis vaccine Bacillus Calmette-Guérin (BCG). This potent bacterial lipid has been shown to induce strong activation of human dendritic cells, marked by upregulation of maturation markers and the secretion of pro-inflammatory cytokines. The relatively simple structure of MMG comprises: 1) a glycerol moiety, and 2) a mycolic acid esterified to one of the primary hydroxyl groups on the glycerol moiety (Figure ). The natural MMG lipids contain either an α-or a keto-mycolic acid, and both types of mycolic acids have comparable immunostimulatory properties. Synthetic MMG analogs can be used for potentiating the immune response induced by subunit vaccines. Their lipid acid moiety is simplified and shorter in comparison to their natural counterparts, yet they possess a comparable activity. Moreover, using a synthetic MMG analog as an alternative to the naturally occurring MMG in vaccines has several benefits, including more simple and scalable synthesis, higher purity, and, in many cases, lower toxicity. Various MMG-analog-containing vaccine adjuvant formulations have been studied, including MMG/dimethyldioctadecylammonium (DDA) liposomes 2,3 combined with Toll-like receptor (TLR) agonists, phytantriol/MMG hexosomes, and an oil-in-water nanoemulsion. The most recent studies have focused on the further development of cationic adjuvant formulation 09 (CAF09), which contains MMG, DDA, and the TLR-3 agonist poly(I:C). The CAF09 formulation contains a specific C 32 MMG analog, MMG-1, which displays a lipid acid stereochemistry that deviates from the native structure. The native structure corresponds to the (2R,3R) lipid acid stereochemistry, whereas the MMG-1 is a 1:1 mixture of (2R,3S)/(2S,3R) configurations (Figure ). In addition to a favorable immunostimulatory effect, MMG-1 has been shown to increase the colloidal stability of DDA liposomes, likely due to increased monolayer hydration and improved lipid packing properties. Interestingly, the lipid acid stereochemistry of different MMG analogs affects their phase behavior and the types of nanostructures the molecules self-assemble into at low temperatures. Analogs with a non-native lipid acid stereochemistry, e.g., MMG-1, assemble into a fully interdigitated subgel phase with a superlattice-like headgroup organization (L c ′ ), while a native-like stereochemistry results in a non-interdigitated gel phase (L β ). 11 Generally, various MMG analogs have been shown to transition directly to the inverted hexagonal (H II ) phase above T m before melting. The transition temperature, however, depends on the configuration and the chain length of the MMG analogs; native-like configurations have a higher propensity for H II formation at lower temperatures. The aforementioned differences appear to correlate with immunostimulatory properties in vitro: neat MMG-1 has been shown to induce more robust immunoactivation and to be less cytotoxic than an otherwise identical analog with a native-like lipid acid stereochemistry. In contrast, in vivo studies with MMG/DDA liposomes suggest that immunoactivity is not influenced by the MMG stereochemistry. Here, we explored the physical mechanisms, which allow MMG-1 to form interdigitated structures at low temperatures. Protrusion of lipid hydrocarbon chains into the opposing leaflet (i.e., interdigitation) is a complex phenomenon that, for other types of lipids, has been shown to occur either spontaneously or to be induced by external factors. These factors include changes in pressure or pH, and exposure to membrane-active proteins or certain small molecules. For MMG analogs with a lipid acid stereochemistry differing from their natural counterparts, the self-assembly into interdigitated structures is a spontaneous process. 11 Various structural features of lipids have previously been shown to correlate with the propensity for spontaneous interdigitation. For example for phospholipids, spontaneous interdigitation is linked to factors that include chain asymmetry, headgroup structure, and positioning, and whether ester or ether linkages are present. While these studies have provided valuable insights into interdigitation specific to phospholipids, the mechanisms of MMG interdigitation may, however, be different.