ScMreB5 ^WT and mutants are well folded. (A) Representative gels of SDS-PAGE profile of purified protein samples of ScMreB5^WT and the mutant constructs. (B) Analytical size-exclusion chromatography using Superdex 200 for ScMreB5^WT (WT); ATPase mutants ScMreB5^D12A, ScMreB5^D70A, ScMreB5^D156A, and ScMreB5^T161A (D12A, D70A, D156A, and T161A); and membrane-binding mutants ScMreB5^I95A, ScMreB5^W96A, ScMreB5^IWA, and ScMreB5^ΔC10A (I95A, W96A, IWA, and ΔC10) in buffer A (300 mM KCl and 50 mM Tris, pH 8.0) shows a single peak corresponding to monomeric ScMreB5, molecular weight ∼38 kD. Peaks (milli absorbance unit [mAU]) corresponding to monomeric protein are marked with asterisk (*). B inset: Calibration curve for size-exclusion chromatography for Superdex 200 using molecular weight standards. The theoretical and estimated molecular weights of ScMreB5^WT monomer are mentioned. (C) Size-exclusion chromatography using Superdex 75 for ScMreB5 ATPase mutant ScMreB5^E134A (E134A); polymerization mutant ScMreB5^K57A (K57A), and ScMreB5^WT (WT) in buffer A (300 mM KCl and 50 mM Tris, pH 8.0) shows a single peak corresponding to monomeric ScMreB5, molecular weight ∼38 kD. Peaks corresponding to monomeric protein are marked with asterisk (*). C inset: Calibration curve for size-exclusion chromatography for Superdex 75 using molecular weight standards. The theoretical and estimated molecular weights of ScMreB5^WT monomer are mentioned. (D and E) Intensity of light scattering measured for ScMreB5^WT and ScMreB5^K57A undergoing polymerization independent of nucleotide addition. Concentration of proteins ScMreB5^WT and ScMreB5^K57A monitored for light scattering are 35 and 5 µM, respectively. Source data are available for this figure: SourceData FS3.