Representative 2D class averages in Figure further highlight the nitrocefin-mediated morphological transformation of the PFK amyloid fibrils. Specifically, while the as-assembled, bare PFK amyloid fibrils feature the parallel β-sheet fiber organization (Figure , two left images) , the nitrocefin-incubated sample yielded striated structures comprising coiled double fibril structure exhibiting a 4.9 Å pitch (Figure , right images). 3D helical reconstruction in Figure , consisting of 507,432 analyzed particles, yields a unique two coiled fibril morphology, with a twist of -7.35º and a rise of ~12 Å, linked together by the embedded nitrocefin molecules. The map resolution achieved (~5 Å, Figure ) does not allow for unambiguous side-chain assignment, presumably due to fiber flexibility and hence inherent heterogeneity between boxed particles. The 90 0 -shifted cross section in Figure highlights the double cross-β structures, capable of binding and burying more than one nitrocefin molecule. At this orientation, each fibrillar-component anchors one nitrocefin molecule, and in total two molecules per unit cell, in which the C-termini lysines (K10-K12) are near the embedded nitrocefin substrate. The helically reconstructed structure in Figure is consistent with the MD-derived structure (i.e., Figure ), in which the positively charged lysine residues face the polar and negatively charged nitrocefin moieties, consequently generating a pronounced hydrophobic surface stabilizing the double layered PFK fibril/nitrocefin assembly.