Extended Data Fig. 3: Cryo-EM insights into I32-10 assembly diversity.

(a) Representative raw micrographs illustrate the I32-10 sample embedded within vitreous ice. The introduction of 100 mM glycine prior to freezing, across three separate instances, effectively mitigated flocculation and aggregation, facilitating downstream data processing to analyze distinct I32-10 assembly states. Movies were acquired across two experimental replicates from samples containing 100 mM glycine for subsequent high-resolution analysis. (b) 3D ab initio reconstructions (C1 symmetry) in cisTEM unveiled a tetrahedral configuration adopted by the majority of I32-10 nanoparticles. (c) Independent C1 3D ab initio reconstructions in CryoSPARC further validated the tetrahedral assembly as the majority I32-10 species. (d) Relion 2D class averages highlighted remarkable heterogeneity in I32-10, encompassing variations in size and geometry. (e) Raw micrographs and subsequent 3D classification in Relion confirmed the existence of diverse off-target and on-target assembly states, consistent with the observations from 2D class averages. (f) An overview of the iterative 3D classification pipeline, as detailed in the Methods section. Supervised 3D classification jobs utilized heavily low-pass-filtered starting models, occasionally leading to the discovery of novel subspecies in output models. Scale bars: (a) 200 nm; (e) 50 nm.