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single cysteine mutants (all with a c-terminal his10 tag) were obtained after site directed mutagenesis  where the natural cys at position 423 had been changed to ser (cysteine-less background). here we follow the individual steps of membrane insertion by single molecule microscopy. first, single cysteine mutants of the pf3 coat protein and of the yidc protein were generated.
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plasmids encoding the single-cysteine mutants of yidc were transformed into the yidc depletion strain mk6s. we focused two lasers in a common detection volume of 10±1 fl and recorded photon bursts from single proteoliposomes with the fluorescent proteins as they diffused through the confocal volume (text s1 and s2).: winterfeld s, ernst s, börsch m, gerken u, kuhn a (2013) real time observation of single membrane protein insertion events by the escherichia coli insertase yidc.