Fig. 2. Time-lapse AFM sequences showing mobile membrane-attached and immobile membrane-inserted mGSDMA3^Nterm oligomers.

a–h A defect-free SLM made from E. coli polar lipid extract was incubated with imaging buffer solution containing 1.5 µM mGSDMA3, which had been beforehand cleaved with 0.4 µM TEV overnight at 37 °C, and imaged in the same solution at 37 °C. Recorded at different time points of the incubation (time stamps indicate minutes), the time-lapse AFM topographs monitor the assembly, disassembly and diffusion of mGSDMA3^Nterm oligomers. From a to h, the topographs follow over the time course different areas of the SLM. The central topographs capture mobile ring-shaped mGSDMA3^Nterm oligomers (indicated by white arrows) which were not there previously (topograph on the left side) and which thereafter disassemble or change position (topograph on the right side). The time-lapse FD-based AFM topographs were recorded in imaging buffer solution at 37 °C (“Methods”), and their full-range color scale corresponds to a vertical scale of 6 nm. Scale bar of 50 nm applies to all topographs. i, Height profiles of mobile mGSDMA3^Nterm oligomers measured along the red lines indicated in the AFM topographs (f, h). Numbers in the upper right corner correlate height profiles to red lines in topographs. Black dashed lines represent the membrane surface (0 nm height). j Maximum heights of ring-shaped oligomers residing in the membrane-attached (MA) and membrane-inserted pre-pore (MI pre) and pore (MI pore) state. Values present mean ± SD and are given in Supplementary Table 1. k Diameters of the maximum height of ring-shaped mGSDMA3^Nterm oligomers residing in the membrane-attached (MA) and membrane-inserted pre-pore (MI pre-pore) and pore (MI pore) state and imaged by time-lapse AFM. Black curves represent Gaussian fits determining the mean ± SD values given. n gives the number of oligomers analyzed. For further information on the representative time-lapse AFM series see Supplementary Fig. 3 and Movie 1.