Lipid Monolayer Reconstitution of FtsA and FtsZ

The two-dimensional lipid monolayers were prepared using E. coli polar lipids (Avanti) as previously described30 (link). Briefly, 0.2 μg of lipids (per well) were floated on storage buffer (lacking EDTA) in a custom-made Teflon block and placed for 1 h in a humid chamber to let the chloroform evaporate. Electron microscopy grids were placed on the monolayers, followed by sequential additions and incubations of 0.1–1 μM FtsA (40 min), 4 mM ATP (20 min), 5 μM FtsZ (5 min) and 4 mM GTP (20 min) in a total volume of 90 μl. The grids were removed and negatively stained with 1% uranyl acetate as previously described28 (link). Grids were inspected and photographed with JEOL 1400 Transmission Electron Microscope coupled with a Gatan Orius CCD camera. Protofilament spacing was measured with boxes or lines using the Plot Profile tool in ImageJ.