The prion [PSI+] is an amyloid isoform of the release factor eRF3 encoded by the SUP35 gene in Saccharomyces cerevisiae yeast. Naturally occurring amyloid complexes have been studied for a long time, yet their structural organization is still not well understood. The formation of amyloid forms of the wild-type Sup35 protein (Sup35p) is directed by its N-terminal portion, which forms a superpleated β-sheet structure. We previously constructed five mutants, each of which carried a replacement in two consecutive amino acids, one in each of the oligopeptide repeats (OR) and in the Sup35p N-terminal region. Mutations sup35-M1 (YQ46-47KK) and sup35-M2 (QQ61-62KK) lead to the compete loss of prion conformation. Three other mutants, i.e., sup35-M3 (QQ70-71KK), sup35-M4 (QQ80-81KK), and sup35-M5 (QQ89-90KK), formed functional prions. In the current study, we investigated the contribution of each mutant peptide to the stability of the prion and aggregation properties, and compared the effects of single mutants and combinations of different mutant alleles. Studies were carried out in yeast strains designed to carry single or a combination of different SUP35 alleles. Based on our analysis, we propose a model that clarifies the 3D organization of the β-sheet within the prion. We also provide evidence that sup35-M2 and sup35-M4 mutations change the 3D structure of prion complexes. We propose that the destabilization of prion complexes in these mutants is due to the decrease of heteroaggregate fragmentation by chaperones.