To better understand the molecular basis for ligand binding, we obtained co-crystals of VRK1-KD (residues Arg3-Glu364) bound to 5 and VRK2-KD bound to 18 (Figure ). Both structures were solved by molecular replacement (Supplementary Table ). VRK1-KD crystals had four protein molecules per asymmetric unit, whereas VRK2-KD crystallized with two copies per asymmetric unit. As observed before, the ligand-bound VRK1-KD and VRK2-KD adopted an active conformation, in which conserved residues from structurally-conserved elements of the kinase domain (Glu83 and Lys71 within αC and the ATPbinding site, respectively -VRK1 numbering) are brought into close proximity and the side chain of Asp197 (VRK1 numbering) from the conserved DFG motif (DYG in the VRKs) points towards the ATP-binding site (Figure In both crystal structures, electron density for the compounds was of good quality and revealed the ligands bound to the protein ATP-binding site, as expected. For VRK2-KD crystals, the ligand could be built in both protein molecules within the asymmetric unit and adopted identical binding modes. In this pose, the compound 2-amino moiety pointed towards the end of the protein ATP-binding site. The 2-amino group and the pyridine N atom of 18 facilitated one hydrogen bond each to the carbonyl and amide groups of VRK2-KD hinge residues Glu122 and Leu124, respectively. In VRK1-KD crystals, the ligand could be observed in three out of the four protein molecules in the asymmetric unit and, surprisingly, was found in two different poses. The first of these was identical to the one observed for LDSM88 bound to VRK2-KD. In the second binding mode, the 2-amino group of 5 pointed towards the solvent and, together with the pyridine N, facilitated HBs with main chain carbonyl and amide, respectively, from VRK1-KD hinge residue Phe134.