Individual SecA molecules were identified and extracted from large-scale AFM images via the Hessian blob algorithm implemented in custom software (Igor Pro 7; Wavemetrics, Portland, OR) (22). A local background level was determined for each individual molecule via Laplace interpolation, providing an accurate baseline from which various molecule metrics were measured. The molecule height was defined as the largest height above the local background level obtained within the molecule. We defined the footprint area as the area occupied by the molecule projected into the flat XY plane, computed as the number of pixels within the molecule scaled by the area per pixel. For kymograph width distribution histograms, line scan traces were analyzed manually. Gaussian fitting was performed using MagicPlot.

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