AFM force spectroscopy on recombinant Cnidoin monomers. (A) Schematic diagram of the AFM force spectroscopy experiment on single Cnidoin molecules, containing several cysteins with thiol groups (orange dots). For breaking disulphide bonds and preventing their reformation, Cnidoin was treated with TCEP and iodoacetamide. (B) Typical force-distance traces of Cnidoin displaying a final double peak, which is characteristic of the cohesin-dockerin rupture . Force-distance traces show characteristic worm-like chain (WLC) behaviour and lack further pronounced features, corroborating the disordered nature of Cnidoin molecules. For quantification of contour and persistence length, a WLC model was fitted to the data (red line). (C) Histogram of the contour length distribution of stretched Cnidoin, yielding an average contour length of 94 nm and a standard deviation of 43 nm. (D) Histogram of the persistence length distribution of Cnidoin, yielding an average persistence length of 0.37 nm and a standard deviation of 0.23 nm. AFM, atomic force microscopy; TCEP, (tris(2-carboxyethyl)phosphine).