Recombinant eADF4(C16) represents an engineered spider silk variant based on the sequence of the core domain of the natural dragline silk protein ADF4 of Araneus diadematus. Previously eADF4(C16) has been shown to self-assemble into cross-p fibrils in a two-step process of nucleus formation and fibril growth. Here, it is shown that structurally converted low molecular weight oligomers can act as nuclei. Further, it could be determined that specifically potassium and phosphate ions strongly influence both nucleus formation as well as fibril growth. Nucleation of fibril assembly could be surpassed by seeding soluble protein with pre-assembled fibrils but also, unexpectedly, with eADF4(C16) sub-micrometer particles. The latter finding reveals that spider silk fibril assembly seems to be rather dependent on the protein sequence than on the structural features, since cross-seeding with other proteins was not possible. (C) 2015 Elsevier Inc. All rights reserved.