Fig. 2: Purified sZipA and FtsZ form co-filament networks in the PURE system.

a Schematic representation of the SLB assays. Purified sZipA-A488 was first incubated on an SLB. The solution on top of the SLB was replaced by a minimal reaction buffer containing 3 µM purified FtsZ-A647 and 2 mM GTP. b Fluorescence images of sZipA-A488 (left) and FtsZ-A647 (right) in the minimal reaction buffer without Ficoll70. c Schematic representation of the SLB assays. Purified sZipA-A488 was first incubated on an SLB. The solution on top of the SLB was replaced by PUREfrex2.0 supplemented with 3 µM purified FtsZ-A647, 2 mM GTP and 50 g L^–1 Ficoll70. d Fluorescence images of sZipA-A488 (left) and FtsZ-A647 (right) in PUREfrex2.0 with Ficoll70. Large-scale filaments with colocalizing FtsZ-A647 and sZipA-A488 are exclusively observed in the presence of Ficoll70. This conclusion is valid in both the minimal reaction buffer and in PUREfrex2.0 background. More fields of view are displayed in Supplementary Fig. 7. e Schematic illustration of the SLB assays with purified FtsZ-A647 (3 µM) and cell-free synthesized ZapA incubated on top of an sZipA-A488-bound SLB. ZapA was expressed from the native gene zapA. f Fluorescence images of sZipA-A488 (left) and FtsZ-A647 (right) in a sample containing cell-free synthesized ZapA and additional 2 mM GTP. Different cytoskeletal network phenotypes were observed when ZapA concentration was increased upon expression of the optimized zapA_opt construct (Supplementary Fig. 9). Scale bars indicate 10 µm.