Structural Analysis of Bacterial Proteins

All purified proteins were precentrifuged for 10 min at 16,000 × g at 4°C. Proteins (0.6 μM each) were incubated with 2 mM GTP and/or 1 mM ATP for 10 min at 37°C in buffer P (Text S1) to observe the structure formation of MsmK and its derivatives. For observing FtsZ polymerization in vitro, mixtures of 0.6 μM FtsZ and/or His-tagged MsmK constructs (0.6 μM each) were incubated at 37°C for 2 min in buffer P to analyze FtsZ polymerization in vitro. Afterward, 2 mM GTP and/or 1 mM ATP was added into the mixtures, which were continuously incubated for 10 min at 37°C. Mixtures were then withdrawn and applied to glow-discharged carbon-coated grids. The grids were air-dried and visualized using an H-7650 TEM (Hitachi, Japan).
The cells in TSB were grown to an OD₆₀₀ of 0.5 and fixed overnight with 2.5% glutaraldehyde at 4°C to observe the bacterial morphology under TEM. For SEM, the cell cultures were grown in TSB to an OD₆₀₀ of 0.5 and spotted onto poly-l-lysine-coated coverslips, followed by washing with PBS buffer. The cells were fixed overnight with 2.5% glutaraldehyde at 4°C. Subsequent dehydration steps were performed using ethanol as previously described (19 (link)). The dried samples were covered with a 10-nm-thick layer of gold and observed with a JSM-6390LV SEM (JEOL, Japan).