Simulated AFM images were produced (fig. S2) by modeling the interaction between a SecA molecule and the AFM tip during the imaging process. The action of “scanning” the AFM tip across a sample surface to produce an image was captured mathematically by the morphological dilation operation, which is responsible for the convolution effect seen in AFM imagery by tips that are not perfectly sharp. To construct a simulated AFM image of SecA, we explicitly computed the morphological dilation between three-dimensional models of SecA and an AFM tip. SecA was modeled via its x-ray crystal structure where each atom was modeled as a sphere with the van der Waals radius (43). The AFM tip was modeled as a cone with a rounded, spherical tip using nominal specifications from the manufacturer (cone angle, 17.5°; tip radius, 8 nm). A simulated AFM image was then produced using custom software (Igor Pro 7; Wavemetrics, Portland, OR). To compare simulated and experimental images (fig. S3), the residual between the images was squared and integrated, with the square root of the result then used to quantify the discrepancy between the images. The discrepancy was normalized as a percentage of the total volume of the experimental image. We then defined the percent agreement as one minus the normalized discrepancy. The agreement was maximized over all relative orientations (translations and rotations) between the experimental and simulated images. To generate equipotential surface maps (fig. S5), electrostatic potentials were calculated with the APBS software (44) at pH 7.

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