AFM-Fold: Rapid reconstruction of protein conformations from AFM images.

High-speed atomic force microscopy (HS-AFM) enables direct visualization of protein dynamics under near-physiological conditions, yet its intrinsic limitation to surface topography prevents atomic-level structural characterization. We present AFM-Fold, a generative AI-based framework that reconstructs three-dimensional protein conformations directly from AFM images. AFM-Fold combines a group-equivariant convolutional neural network, which extracts low-dimensional collective variables (CVs) from AFM images, with a guided diffusion process that generates conformations consistent with the inferred CVs. Using pseudo-AFM images of adenylate kinase, AFM-Fold accurately reproduced not only the open and closed conformations, but also a continuous range of intermediate conformations spanning the open-closed transition. Application to 159 experimental HS-AFM frames of the flagellar protein FlhAC further demonstrated that AFM-Fold yields conformations more consistent with experimental images than rigid-body fitting of the crystal structure, and it captures time-correlated domain motions that reflect underlying conformational dynamics. AFM-Fold enables rapid, physically plausible structure estimation from individual AFM images, typically within 1 min per frame, without relying on molecular dynamics simulations. This unified and computationally efficient pipeline opens a route to high-throughput structural analysis of HS-AFM movies.