Biofilm-protected Pseudomonas aeruginosa underlies stubborn, treatment-refractory infections. FapC, the major biofilm-forming functional amyloid, is essential for matrix integrity, yet how it progresses from a disordered monomer to a mature fibril has remained unresolved. We present a continuous, atomistic picture of FapC biogenesis by integrating solution NMR, single-particle cryo-EM, and all-atom molecular dynamics (MD). NMR ensemble analysis defines the conformational landscape and residual structure of monomeric FapC. A high-resolution cryo-EM density map allowed model building by using AF FapC model that reveals an irregular, triple-layer β-solenoid cross-β architecture formed by a single protofilament. MD simulations initiated from monomeric and fibrillar states connect these endpoints, identifying sequence-encoded nucleation motifs, registry-locking contacts across layers, and solvent-exposed segments that modulate oligomer stability. Perturbation simulations suggest routes for disassembly and highlight interaction hotspots whose disruption destabilizes the cross-β core. Together, these results rationalize how FapC funnels conformational heterogeneity toward a β-solenoid fold, how interstrand and interlayer interactions consolidate fibril stability, and why the resulting scaffold is resilient in the biofilm environment. By establishing both structure and mechanism, our work provides testable hypotheses for targeting functional amyloid assembly in bacteria and offers design principles for inhibitors aimed at weakening biofilm integrity and combating antimicrobial resistance.
Ümit Akbey (Sun,) studied this question.