The selected XChem hit analogues were screened using X-ray crystallography (using the PaFabF C164Q variant) and the BLI assay (against PaFabF C164A). In the BLI assay, some compounds showed no binding and some super-stoichiometric binding, but for none of them binding constants could be determined (Table ). Using X-ray crystallography, four analogues were found to bind to FabF (compounds 53, 54, 56, 107, Table , Figure ). Analogues 54 and 107 were found to bind in the active site of FabF with a similar binding pose as the original hit compound 21 (Figure ). In contrast, the analogue 53 bound to the surface of the protein which might indicate that the cyclopentane moiety is too large to be accommodate in the active site. Analogue 56 was also found to bind into the active site albeit with a different binding mode compared to the parent fragment 21 (Figure ). The hydroxyl group of fragment hit 21 established direct hydrogen bonds with the catalytic His304 and His341 residues whereas the hydroxyl group of analogue 56 engaged in hydrogen bonding with Thr306 and Thr308 instead. At the same time, analogue 56 formed hydrogen bonds with four water molecules acting as hydrogen-bond bridges between the ligand and protein residues (Asp266, Ala280, Gly279, Glu315, Thr306, Gly311, His304 and His341). Furthermore, the amide group is engaged in stacking interactions with the aromatic ring of Phe398 (4.2/4.4 Å distance between the C and O atoms, respectively and the centre of the phenyl ring).