The purified darobactins D22, D23, D31, D32, D36, D37 and D38 were structurally verified via NMR (Figure S 46-75, Table S 8-13) and tested against a panel of different Gram-negative pathogens including A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. The activity was compared to previously published DA activity data and the crude extract data shown above (Table ). Darobactins are not active against pathogens with phylogenetically distant BamA gene loci, like Proteus mirabilis or Serratia marcescens (Table ; Figure S 42), as expected. However, many new derivatives depict promising antibacterial activity against the majority of tested pathogens (Table ) and are in line with the previously generated MIC data of crude extracts (Table ). Compounds D22 and D31 stand out by demonstrating up to an eight-fold increased activity against the panel of pathogens compared to native DA. Additionally, there was up to a two-fold improved activity compared to D9, most interestingly against Acinetobacter strains. Moreover, direct comparison of D22, D31 and D32 emphasises the minor influence of manipulations made in core sequence corresponding to amino acid position 4. The substantial influence of changes to position 6 is reflected by comparing the differing activities of D22 and D38 in addition to the activities of D9 and D37. Changing L-serine to L-threonine or L-arginine to L-threonine decreased bactericidal activity significantly (Table ), whereas a basic sidechain can enhance the activity demonstrated by comparison of DA with D36. However, these effects are generally species-specific, most notably for the K. pneumoniae strains. Furthermore, no cytotoxicity was observed against the human cell line HepG2 as published for DA and D9. To investigate the SAR of D22, which exhibited the best activity profile of all known darobactins, a cryo-EM co-structure determination of D22 bound BAM complex was initiated to study the potentially changed mode of binding (MoB). The structural basis of superior activity of D22 was analysed by single-particle co-structure elucidation via cryo-EM of E. coli BAM-D22 complex (Figure ) and compared to the co-structure of BAM-D9 (Figure ). The cryo-EM structure of the E. coli BAM complex was resolved with an overall resolution of Q441 from β-strand 2 is formed due to its side-chain remodeling (Figure ). These conformational changes are likely to be responsible for increased binding affinity and consequently improved bioactivity at least in A. baumannii strains. Furthermore, the significantly changed interaction and orientation at the C-terminal end is not at the expense of one of three N-terminal hydrogen bond interactions, maintaining proper closing of the lateral gate of BAM (Figure 3 c, d). The fact that T4 is not involved in direct binding with BamA β-barrels makes this residue attractive for evaluating other amino acids or other moieties for improving pharmacological properties. Taken together, the different darobactin-BamA binding interactions of DA, D9, and D22 likely reveal the structural basis for enhanced activity of engineered darobactins, which are more closely paired with BamA to mimic the β-sheet prolongation and to lock the BAM complex in the inactive state by sealing the lateral gate tightly (Figure , 3). These structure-activity based findings are reinforced by the results of analysing the different binding kinetics for DA and D9 versus D22 on purified E. coli BamA revealing KD values of 0.278 µM, 0.3 µM and 0.159 µM, respectively (Figure S 44). Nevertheless, more extensive activity profiling assays must be performed to generate a more valid correlation between the increased activity of D22 and the structural changes compared to DA and D9. As D22 distinguishes itself by its superior activity, especially against tested Acinetobacter strains (Table ), this derivative was tested in a MIC assay using hard-to-treat clinical isolates of carbapenem-resistant Acinetobacter baumannii (CRAB), where most of the commonly used antibiotics are not active (Table ).