Cryo-EM methods to study binding between amyloid fibrils and chemical compounds.

Amyloid fibrils formed by amyloid proteins such as α-synuclein (α-syn), Amyloid-β (Aβ) and Tau are central to the pathology of neurodegenerative diseases like Parkinson's disease (PD) and Alzheimer's disease (AD). Structural elucidation of fibril-ligand interactions is essential for the rational design of imaging probes and therapeutic inhibitors targeting these pathological aggregates. Here, we present a comprehensive cryo-electron microscopy (cryo-EM)-based workflow for modeling small-molecule binding to amyloid fibrils, with a focus on α-syn-ligand complexes. The protocol integrates optimized fibril sample preparation, helical reconstruction, and iterative 2D/3D classification to yield high-resolution density maps suitable for atomic modeling. Ligands are incorporated by generating coordinates from SMILES strings and restraint files from Phenix eLBOW, followed by manual docking and real-space refinement. Using CCA-α-syn complex as a case study, we demonstrate precise ligand placement into specific fibril binding sites (the C-pocket, N-pocket, and a back-surface groove of the fibril core distinct from typical globular protein pockets). Subsequent structural refinement preserved key interaction features, including π-π stacking and side-chain hydrogen bonding. Validation metrics confirm the stereochemical integrity and good model-to-map fit of the final fibril-ligand complex structures. Overall, this workflow enables accurate modeling of ligand engagement with amyloids even at ∼3-4 Å resolution and provides a scalable framework for structure-guided ligand discovery in neurodegenerative disease research.