Fig. 2. CmΔZntA homology model, purification, reconstitution in proteoliposomes, and metal-dependent ATPase activity. (a) 3D homology model of CmΔZntA_166–794 predicted with Robetta Protein Structure Prediction Server (http://robetta.bakerlab.org/; M²⁺ = Zn²⁺. Cd²⁺, Hg²⁺, Pb²⁺) and (b) close-up view of the putative transmembrane metal binding site involved in substrate selection. Cys438, Cys440 (on M4) and Asp777 (on M6) create the putative high affinity binding site conserved in ZntAs, and Lys756 (on M5) acts as a built-in counterion. (c) Size exclusion chromatogram of purified CmΔZntA in Cymal-7 micelles. (d) Corresponding SDS-PAGE gel of CmΔZntA and SDS-PAGE analysis of CmΔZntA incorporation in proteoliposomes (PL) vs. control (Cont.) liposomes (P: proteoliposomes/liposomes pellet isolated by ultracentrifugation; S: corresponding supernatant). (e) Dynamic light scattering (DLS) analysis of control liposomes and CmΔZntA proteoliposomes characterizing the average SUV diameter (in nm) and size distribution. (f) Relative metal-stimulated CmΔZntA ATPase activity in the presence of different metals in Cymal-7 micelles (metal concentration = 50 μM). (g) Relative metal-stimulated ATPase activity in the presence of different metals for CmΔZntA reconstituted in proteoliposomes (metal concentration = 50 μM). All data are mean ± s.d. (n = 3).