Fig. 1: In vitro assembly analysis indicates cooperativity in most regimes.

a–b Schematics of in vitro assembly and representative electron micrographs of in vitro-assembled nanomaterials. Scale bars, 50 nm. c Possible dependence of assembly structures (as Schlegel diagrams; transparent panes, voids) on initial subunit-stoichiometry. d, j Representative SEC traces from in vitro assembly reactions. Assembled nanomaterials and residual unassembled components were quantified by peak area (green, I53-40; purple, I53-50; orange, unassembled components). Green, purple, and orange bars indicate the fractions pooled for subsequent analyses. e, k Cooperativity analyses, in which peak area (normalized to the theoretical yield, gray dashed lines) is plotted against initial subunit stoichiometry. Data from reaction sets with variable [T]₀ or [P]₀ are shown using triangular or pentagonal symbols, respectively; the variable:constant component molar ratio is shown above the x-axis. Error bars represent the standard deviation of three technical replicates, and peak area is normalized to the average total signal obtained from six 50 μM equimolar assemblies. A characteristic chevron-like feature emerges when grouping assembly reactions initiated with sub-stoichiometric (dotted gray lines) or super-stoichiometric (solid gray lines) concentrations of pentamer. f, l SDS-PAGE analysis of nanomaterials assembled in vitro or in vivo (*) by co-expression in E. coli. Initial subunit ratios are indicated by colored bars ([T], blue; [I53-40P], yellow; [I53-50P], red); 25 kDa, 20 kDa, and 15 kDa marker locations are indicated by black arrows. g, m Analysis of the post-assembly T:P ratio in SEC-purified I53-40 (green) and I53-50 (purple) nanomaterials by PAGE gel densitometry. Data are normalized to the average ratio of signals (dashed line) in samples confirmed to be complete by native MS. Markers are shaded according to increasing initial [T]₀:[P]₀ ratio. h, n Non-denaturing PAGE of SEC-purified nanomaterials. i, o SDS-PAGE analysis of SEC-purified residual unassembled component fractions; 25 kDa, 20 kDa, and 15 kDa marker locations are indicated by black arrows. The datasets corresponding to equimolar ([T]₀:[P]₀ = 1) assembly reactions of I53-40 (f–i) and I53-50 (l–o) are enclosed in black boxes. Source data are provided as a Source Data file.