Figure : Overview of the workflow of Φ-value analysis of amyloid fibril elongation. 1: Identify aliphatic residues viable for hydrophobic deletion, such that all distinct structural elements of the fibril are probed. 2: Relative elongation rates of all mutants are investigated by Quartz Crystal Microbalance (QCM), by incubating immobilized wildtype (WT) fibrils with both WT and variant monomer. WT monomer is demonstrated to maintain elongation rates after elongation by variant protein, demonstrating fibril structure is faithfully propagated. 3: Chemical denaturation series are performed on all variants capable of propagating the WT fibril structure. Variant protein is seeded with small proportions of WT fibrils to induce the correct fibril structure in the mutant protein. The denaturation series are globally fitted with the cooperative polymerization model. Mutant fibrils are imaged by AFM to demonstrate that overall fibril morphology is maintained. 4: Calculated Φ-values are used to constrain MD-simulations of the TSE that are started from the fibrillar state. 5: Use FoldX to calculate the ∆∆G ‡-A from the simulated TSE structures to check if the simulation captures the experimental kinetic data. Measure depolymerization kinetics of the WT fibrils from a denaturation series, calculate the m-value and the Tanford value. Compare this to the simulated TSE to further comfirm the plausibility of the simulated TSE.