Fig. 6: Cryo-ET reveals severe membrane wobbling in E. coli ED1a cells treated with TET.

a Ion beam image of the E. coli cells selected for FIB-milling. Scale bar 20 µm. For each strain, cell batches for FIB milling were prepared in three biological replicates from independent cell cultures grown from individual colonies. Cryo-EM grids for each sample were prepared in at least two technical replicates. b Scanning electron beam image of the grid square with FIB-milled lamella of bacterial cells. Scale bar 30 µm. For each cryo EM grid, at least three lamellae were prepared, comprising 5−15 individual cells per lamella. c Transmission electron microscopy (TEM) image map of a lamella. The black targets point to the regions of subsequent tilt series data collection. Scale bar 4 µm. For each obtained lamella, maximum number of tilt series were collected, excluding pre-selection of cells. d Digital slices from the reconstructed tomograms, demonstrating the membranes wobbling in E. coli ED1a cells treated with 8 μg/ml (2X MIC) TET for 30 min (right-most panel). Scale bar 0.2 μm. T1: timepoint 1 (30 min LB), T2: timepoint 2 (30 min TET). e Digital slices at different z-heights from tomograms of various E. coli ED1a cells treated with TET for 30 min. The images were low pass filtered to better visualize the membrane wobbling. Scale bars 0.2 μm. f Intermembrane distance in the cells used for cryo-ET and subtomogram averaging. The violin plot shows the smoothed density of every measurement (gray dots), the first and third quartiles (black solid lines), and the median (red solid line); n represents the number of points at which measurements were made for 10 K-12 (LB), 10 K-12 (TET), 10 ED1a (LB), and 7 ED1a (TET) cells. The violin plots for each of these cells individually is shown in Supplementary Fig. 4a−d. The schematic cartoon of intermembrane distance analysis is shown in Supplementary Fig. 4e. The statistical significance was calculated using a two-sided unpaired t test. The significance symbols indicate P values: ns for P = 0.6758 **** for P < 0.001. Source data are provided as a Source Data file.