Second-site suppressor libraries of the parent inactive mutants V62Q, M66L, M66S and V68G were constructed by individually randomizing each of the partners in both the NMR and X-ray structures of the protein. Suppressors were identified by screening on selective media at decreasing NaCl concentrations in an E. coli strain deleted for dgkA. (A,B), (D,E), (G,H) and (J,K) show the phenotype of the suppressors isolated from the libraries of V62Q, M66L, M66S and V68G, respectively at (A, D, G, J) 0.15% NaCl, 0.01% arabinose, (B,K) 0%NaCl, 0.01% arabinose and (E, H) 0.03% NaCl, 0.01% arabinose. (C, F, I, L) Representative plate photograph showing the location of the variants and the controls for the screening of suppressors of the parent inactive mutants V62Q (A,B), M66L (D,E), M66S (G,H) and V68G (J,K), respectively. '(X)' and '(N)' indicate the structure from which the residue partner has been chosen that is either X-ray or NMR structure, respectively. Parent inactive mutant, Wild-Type (WT) DgkA and the empty vector pBAD-33 act as reference, positive and negative controls, respectively. The true suppressors are anticipated to appear before the parent inactive mutant on the plate when grown in low salt conditions. Suppressors (V62Q, A41G), (M66L, V38A), (M66S, G35A), (V68G, A100V), which are in spatial proximity in the crystal structure restore the growth defect of the parent inactive mutants. A similar analysis for the parent inactive mutant I67V has been shown in Figure 6C–E. Overall, suppressors were only found at positions proximal to the parent inactive mutant in the X-ray structure of DgkA.