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 imaging applications. AFM is capable of producing detailed three-dimensional topographical height images with a high signal-to-noise ratio. This is a key capability of AFM, which enables the structural features of individual molecules to be studied without the need for ensemble averaging, and could offer structural analysis applications where heterogeneity of molecular populations, structural variations between individual molecules, or population distribution properties in general, hold important information. Here, a software tool called Trace\_y designed to reconstruct the three-dimensional (3D) surface envelopes of individual helical filament structures from topographical AFM height 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 from a single amino-acid sequence due to structural polymorphism is not fully understood. The software and the workflow presented here allows the structural information encoded in topographical AFM height images to be extracted and understood as 3D contact point clouds. This approach will facilitate the use of AFM in structural biology, in integrated methodologies together with other structural analysis tools to understand the structures and behaviours at individual molecule level. ### Competing Interest Statement The authors have declared no competing interest.