RESUMEN
Atomic force microscopy has emerged as a valuable complementary technique in membrane structural biology. The apparatus is capable of probing individual membrane proteins in fluid lipid bilayers at room temperature with spatial resolution at the molecular length scale. Protein conformational dynamics are accessible over a range of biologically relevant timescales. This chapter presents methodology our group uses to achieve robust AFM image data of the General Secretory system, the primary pathway of protein export from the cytoplasm to the periplasm of E. coli. Emphasis is given to measuring and maintaining biochemical activity and to objective AFM image processing methods. For example, the biochemical assays can be used to determine chemomechanical coupling efficiency of surface adsorbed translocases. The Hessian blob algorithm and its extension to nonlocalized linear features, the line detection algorithm, provide automated feature delineations. Many of the methods discussed here can be applied to other membrane protein systems of interest.