<p dir="ltr">The obligate human pathogen, <i>Neisseria gonorrhoeae </i>(Ngo), has continued to acquire widespread antibiotic resistance. Ngo is the causative agent of the sexually transmitted disease gonorrhea, and can cause additional complications such as endocarditis, septicemia, and infertility if left untreated. The Centers for Disease Control and Prevention (CDC) now recommends a treatment option of a single drug of last resort, ceftriaxone, leaving a need for novel therapeutics against this pathogen.</p><p dir="ltr">Like many bacterial pathogens, Ngo is Gram-negative consisting of both an inner membrane (IM) and outer membrane (OM). The transmembrane proteins in the IM have primarily an α-helical fold, while the transmembrane proteins in the OM have a β-barrel fold. These β-barrel outer membrane proteins (OMPs) have essential functions in regulating the homeostasis and nutrient acquisition of the cell, in addition to promoting virulence in pathogenic strains. These OMPs are folded and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. In <i>E. coli,</i> BAM consists of five proteins: BamA, an OMP itself, and four lipoproteins, BamB, C, D, and E.</p><p dir="ltr">Here we present our work toward the structural characterization of BAM from Ngo (<i>Ng</i>BAM) using cryo-EM. Ngo lack a homolog of BamB and may function as a four component complex. To better understand the mechanism for how <i>Ng</i>BAM is able to mediate OMP biogenesis despite lacking a component that is critical in <i>E. coli</i>, we determined the cryo-EM structure of <i>Ng</i>BAM, which revealed several distinct features including that the barrel domain of BamA being observed in the inward-open conformation. We also investigated <i>Ng</i>BAM as a therapeutic target, by studying its interaction with a novel broad spectrum antibiotic darobactin. We first showed darobactin is effective against the laboratory strains of NgoFA19 and ATCC-49226. We also show it is effective against the human isolate WHOX, with a comparable MIC to ceftriaxone. To structurally characterize the mechanism of inhibition by darobactin, we used cryo-EM to determine the structures of <i>Ng</i>BAM bound to two darobactin compounds. In these structures, darobactin binding was accompanied by large conformational changes in <i>Ng</i>BamA. To further probe the effects of darobactin on the conformational plasticity of <i>Ng</i>BAM we performed experiments using double electron-electron resonance spectroscopy, which showed distance changes between the engineered site labels consistent with the conformational changes observed in our structural observation. In addition, narrowing of the peak distributions indicated that darobactin binding was reducing the overall conformational heterogeneity of the complex. Taken together, the work presented here contributes to the understanding of how <i>Ng</i>BAM functions in folding and inserting OMPs and provides a foundation for future structure based drug design of darobactin and other potential compounds.</p>