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<b>Fig A. Cryo-EM processing workflow in Cryosparc.</b> (A) Raw micrograph processing on CryoSPARC and steps required to proceed with 2D classification. (B) 2D classes of selected particles. (C) <i>Ab initio</i> reconstruction of cryo-EM density using Icosahedral symmetry. (D) Homogeneous refinement of map that resulted in 3.15 Å resolution. The final density map obtained can be viewed in cyan through its 5-fold and 2-fold symmetry axes. (E) Quality assessment metrics. (upper left) GSFSC resolution as computed from the processing software CryoSPARC, indicates a final map resolution of 3.15 Å resolution. (upper right) Local resolution of the final map plotted on the surface of the map as a colour gradient that ranges from 2.8–3.8 Å. (lower left) Euler angle distribution plot that shows the angular distribution of the particles (lower right) Model vs FSC plot evaluates the map-to-model fit match at 0.5 threshold. <b>Related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012873#ppat.1012873.g002" target="_blank">Fig 2</a>. Fig B. Evaluation of the increased density at the center of the VLPs.</b> (A) Comparison of the cryo-EM density of the current study VLP (blue), EMD_6829 (yellow), EMD_6752 (green) and EMD_14186 (purple), shown from the face of the 5-fold axis, (1st column), the 2-fold axis (2^nd column), and the central slice of each particle (3^rd column). (B) Graphical plot of the normalized radial intensity of each particle density plotted against the radius (Å). (C) Comparison of the pdb models of the ASUs of the VLP originating from this study (blue), 5yhq (yellow), and 5xs5 (green) rotated along the x axis by 90° from left to right. <b>Related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012873#ppat.1012873.g002" target="_blank">Fig 2</a>. Fig C. LCMS analysis assessing the cleavage of the viral protein VP0 as indicator of viral maturation.</b> (A) Full VP0 amino acid sequence, with VP4 and VP2 indicated in red and green, respectively. Bold and underlined indicates the concatenated VP4/VP2 sequence of the proteotypic peptide for VP0 upon digestion. For VLPs that underwent viral maturation, the C-terminal proteotypic peptide of VP4 (EVAAPLQ) and the N-terminal proteotypic peptide of VP2 (SPSVEACGYSDR) should be identified, while for immature VLPs, the concatenated sequence of these VP4-VP2 termini (EVAAPLQSPSVEACGYSDR) should be identified. (B) MS analysis revealed the presence of VP0 (concatenated VP4-VP2) in high abundance. Additionally, the C-terminal and N-terminal proteotypic peptides of VP4 and VP2, respectively, were identified, however in extremely low concentrations (~3 orders of magnitude lower than that of VP0). <b>Related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012873#ppat.1012873.g002" target="_blank">Fig 2</a>. Fig D. AFM nanoindentation results for EV71, CVA6 virions and VLPs.</b> (A) Corrected height of the particles (EV71 VLPs (black), EV71 virions (red), CVA6 VLPs (blue), and CVA6 virions (violet)) as determined by AFM measurements. (B) Viral spring constant (N/m). (C) Critical force (nN). (D) Critical indentation (nm). P values are indicated by asterisks: p<0.001 (***), p<0.01 (**), and p<0.05 (*). N.s.: not significant. The CVA6 data is presented again for direct comparison with the EV71 data. <b>Related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012873#ppat.1012873.g004" target="_blank">Fig 4</a>. Fig E. AFM particle stability parameters not affected by formaldehyde inactivation.</b> To investigate whether stabilizing effects of formaldehyde treatment accounted for the observed differences in the mechanical properties between virions and VLPs, a supplementary batch of CVA6 VLPs underwent the same formaldehyde treatment as the CVA6 virions, after which we again performed nanoindentation force spectroscopy experiments using the AFM. (A) Viral spring constant (N/m). CVA6 VLP (blue), CVA6 inactivated virions (violet), and CVA6 inactivated VLPs (green). (B) Critical force (nN). (C) Critical indentation (nm). P values are indicated by asterisks: p<0.001 (***), p<0.01 (**), and p<0.05 (*). N.s.: not significant. From these experiments, we concluded from this data that there was no significant difference between treated and untreated CVA6 VLPs. The same is assumed to hold true for EV71 VLPs. Therefore, it was concluded that the inactivation process did not have any additional stabilizing effects on the CVA6 virion capsids. <b>Related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012873#ppat.1012873.g004" target="_blank">Fig 4</a>. Figure F. Mass photometry data on CVA6 VLPs.</b> To further investigate the content of the CVA6 VLPs, mass photometry experiments were performed. The results revealed a widespread distribution, indicating heterogeneity in the VLP content. Four populations were identified (red Gaussian fits) which approximately correlated with the molecular weights of the empty particle (~3.8 MDa) and particles containing vRNA fragments coding for proteins 3C (~0.34 MDa; Gaussian peak at 4.2 MDa), 3D (~0.86 MDa; Gaussian peak at 4.6 MDa), and the P1 region (~1.59 MDa; Gaussian peak at 5.1 MDa). Differences observed for the P1 peak may have arisen from the Gaussian fits applied to the data and the limited statistical sample size. Table A. Measured size and determined mechanical parameters for the different capsid structures. Related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012873#ppat.1012873.g004" target="_blank">Fig 4</a>. Table B. Deduction of Young’s moduli from measured size parameters for the different capsid structures. Related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012873#ppat.1012873.g004" target="_blank">Fig 4</a>. Table C. Processing and structural information of our CVA6 VLP. (DOCX)