The hit-and-run of cell wall synthesis: LpoB transiently binds and activates PBP1b through a conserved allosteric switch

This repository contains single-molecule data and Matlab analysis code related to the manuscript "The hit-and-run of cell wall synthesis: LpoB transiently binds and activates PBP1b through a conserved allosteric switch" (Figures 1, 2, 4, 5).

1) smFRET data in Figures 1 and 2 were analyzed with ebFRET software (van den Meent et al. Biophys J 2014), using outputs from a previously published raw movie processing pipeline (Graham et al. Mol Cell 2016). The resulting data (included here) were further parsed with custom Matlab code published in Gilman, Shlosman & Same Guerra et al. bioRxiv 2024 and publicly available on Zenodo (https://zenodo.org/records/13910538) to obtain statistics and fits of population histograms, dwell times and transition rates.

2) smFRET data in Figure 4 were processed with the publicly available COSMOS pipeline (Smith et al. Nat Commun 2019) to obtain trajectories of donor and acceptor colocalization events. These data were further analyzed using custom Matlab code (BindingFRETAnalysis.m) to filter out spurious events and obtain statistics and fits of association rates and dwell times of binding.

3) Single-particle tracking data in Figure 5 were parsed with the TrackMate plugin in ImageJ (Ershov et al. Nature Methods 2022) to obtain .xml track files (example datasets included here), which were further analyzed either with Spot-ON tracking software (Hansen & Woringer Elife 2018) to quantify the populations and diffusion coefficients of free vs immobile particles or with custom Matlab code (CellDwellTime.m) to quantify the dwell times of those immobilization events.