Figure 1

Automated DNA bend angle analyses at protein-DNA complex sites. (a) Schematic representation of DNA bend angle measurement at the site of a bound protein in AFM image: the DNA bend angle Θ at the position of a bound protein (arrow) is determined from the angle Φ measured by tangent overlay as indicated: Θ = 180° − Φ. (b) Protein peaks (arrows) are selected by thresholding and their coordinates are obtained (lower part in (b), arrows). (c, d) DNA fragments are identified by thresholding and distinguished from protein signals by shape filtering (c). Selected DNA filaments are then skeletonized (d). (e) The DNA skeleton lines are fed into MatLab together with protein position coordinates. In the resulting overlay images, cyan circles indicate the protein positions and color lines the DNA fragments. Protein peaks located at DNA fragment ends (grey arrows in (b)) are discarded. The angle Φ between tangent lines to points at a user defined distance (here: 8 nm) from the protein positions on DNA is automatically measured for all protein-DNA complexes, and the corresponding DNA bend angles Θ are returned by the software. Scale bars: (a) 200 nm, (e) 500 nm.