Negative Staining of RodA:PBP2 Complex

For negative staining, 2.5 μl protein solution consisting of RodA:PBP2 complex at a concentration of 0.005 mg ml⁻¹ in 0.01% DDM (w/v) was added to a glow-discharged carbon-coated copper grid (Electron Microscopy Science) and allowed to adsorb for 30 s. Grids were then washed twice with deionized water and stained twice with freshly prepared 1.5% (w/v) uranyl formate. Filter paper was applied to the grid to absorb residual liquid between each step. Samples were then allowed to dry for approximately 2 min. Images used to generate the 2D classes shown in Figure 3 were collected at room temperature using a Philips Tecnai T12 electron microscope equipped with an LaB6 filament and operated at 120 kV. Images were collected at a magnification of 67,000-fold, corresponding to a pixel size of 1.68 Å, and a defocus value of −1.5 μm on a Gatan 4K CCD camera using a low-dose collection procedure. Approximately 1000 particles were manually picked in Relion^{36 (link)}, then an auto-picking routine picked the remaining particles for a total of 32,152. Two-dimensional class averages were calculated with Relion. Similar class averages were observed when calculated with EMAN2^{37 (link)}.