To record an insertion and folding event of a single LacY polypeptide, we had to approach and withdraw the AFM tip to and from proteoliposomes frequently. For example, in >137,000 attempts approaching the AFM tip to proteoliposomes containing SecYEG, we detected only 172 events, showing that the LacY polypeptide inserted and folded one or more structural segments into the lipid bilayer (at a folding time of 1 s). Similarly, we needed >193,000 attempts to detect 155 insertion and folding events of the LacY polypeptide in the presence of YidC at a folding time of 1 s. These numbers do not represent efficiencies of insertion because, in each attempt, an unfolded LacY polypeptide is approached to the surface of a proteoliposome showing relatively low densities of translocases and/or insertases (figs. S1E and S2, D and E). Under these conditions, the probability that the unfolded LacY polypeptide interacts with a translocase and/or insertase is rather low. In addition, because the polypeptide had been attached to the AFM tip unspecifically and transiently, it slipped quite often from the tip (1214).

Datasets of 478 (in the presence of YidC), 395 (in the presence of SecYEG), 397 (in the presence of SecYEG-YidC construct), and 313 (in the presence of SecYEG and YidC from the cleaved SecYEG-YidC fusion construct) force-distance curves, each recording a folding event, were analyzed. Probability distributions were compared with the uniform distribution using χ2 tests. Two-tailed Z tests were performed to evaluate the significance of difference between insertion probabilities for YidC, SecYEG, and SecYEG-YidC construct. Analysis of covariance was used to test the difference between the slopes for kinetic measurements. The difference between groups and distributions was considered to be statistically not significant when P > 0.05 and statistically significant when *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars give SE. Statistical analysis was accomplished in R and Prism 7.

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