Trace_y: Software algorithms for structural analysis of individual helical filaments by three-dimensional contact point reconstruction atomic force microscopy.

Atomic force microscopy (AFM) is a powerful and increasingly accessible technology that has a wide range of bio-imaging applications. AFM is capable of producing detailed three-dimensional topographical images with high signal-to-noise ratio, which enables the structural features of individual molecules to be studied without the need for ensemble averaging. Here, a software tool Trace_y, designed to reconstruct the three-dimensional surface envelopes of individual helical filament structures from topographical AFM images, is presented. Workflow using Trace_y is demonstrated on the structural analysis of individual helical amyloid protein fibrils where the assembly mechanism of heterogeneous, complex and diverse fibril populations due to structural polymorphism is not understood. The algorithms presented here allow structural information encoded in topographical AFM height images to be extracted and understood as three-dimensional (3D) contact point clouds. This approach will facilitate the use of AFM in structural biology to understand molecular structures and behaviors at individual molecule level.