Fig. 8: Function-structure analysis of RipC activation.

a PG hydrolysis activity of RipC under different conditions. Buffer solution in the presence ATP-Mg²⁺ was used as negative control and the PaeFtsEX/EnvC/AmiB as positive control. Only in the presence of ATP-Mg²⁺ or trapped at a post-hydrolysis state by ATP-Mg²⁺/vanadate, MtbFtsEX/RipC showed minimal PG hydrolysis activity. n = 3 replicates, error bars were presented as mean ± SD. Two-tailed unpaired t-test was applied; **P < 0.005, n.s. no significant difference. The P-values are shown in figure and source data are provided as a Source Data file. b, c Cryo-EM density map of the ATP-activated RipC-bound MtbFtsEX complex is shown in grey with 60% transparency surface with two conformations, Type 1 (b) and Type 2 (c). In the Type 1 state, there is a weak density of NlpC/P60 domain, while in the Type 2 state, the corresponding density completely disappears. The model for the Type 1 conformation is shown in blue, and the model for the Type 2 conformation is shown in the red. d Side-view of superposition of the RipC-bound MtbFtsEX complex in three different states: ATP-free (in yellow), ATP-activated Type 1 (in blue), and ATP-activated Type 2 (in red); the tilt of RipC upon ATP hydrolysis is highlighted. The FtsEX components are shown as transparent surfaces, and the RipC hydrolase is represented in licorice. e, Upper: Front-view of the overlay of the RipC-bound MtbFtsEX complex in three different states: ATP-free (in yellow), ATP-activated Type 1 (in blue), and ATP-activated Type 2 (in red); Lower: conformational changes of FtsE upon ATP binding. f, g Conformational changes in ECD (f) and RipC (g) observed during the transition from ATP-free state to ATP-activated Type 1 state. The ECD domains are shown in ribbon, while RipC is represented in transparent ribbon (70%). Structural differences are labeled, with the ATP-free state in yellow, and the ATP-activated Type 1 state in blue. h, i Conformational changes in ECD (h) and RipC (i) were observed during the transition from ATP-activated Type 1 state to ATP-activated Type 2 state. Structural differences are labelled, with the ATP-activated Type 1 state in blue, and the ATP-activated Type 2 state in red.