The worm-like chain (WLC) model was fitted to every unfolding force peak in each force-distance curve recorded upon unfolding individual membrane protein (13). A persistence length of 0.4 nm and contour length of 0.36 nm per amino acid were used for data analysis to reveal the contour length (number of amino acid) of the polypeptide unfolded and stretched in each detected force peak. Contour length histograms of all force peaks detected in all unfolding force-distance curves were fitted with a Gaussian mixture model, which revealed 10 force peak classes for LacY (17). The 10 force peak classes were mapped to the secondary structure of LacY (Fig. 1D) taking into account the polyGly and His8-tag elongation of the C-terminal end. If the force peak class located the beginning/end of a stabilizing structural segment on the mica-facing side of the membrane or within the lipid membrane, then the thickness of the membrane was considered for peak mapping (13).

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