Supplementary Figure 4

(A) Snapshots showing ‘cluster’ formation embedded in vitreous ice for cryo-EM observation. The centers of microtubule nucleation clusters have high densities of microtubules. (B) Fourier Shell Correlation (FSC) of the 3D reconstruction of the SSNA1-microtubule complex. The global resolution of the 3D reconstruction is 6.1 Å according to the FSC = 0.143 criteria, though it is only effective for the core of tubulin according to the local resolution mapping shown in D. (C and D) Local resolution representation of the 3D reconstruction according to the resolution-color code on the right color bar. (C) with a threshold that includes the SSNA1 decoration and (D) with the threshold that allows the visualization of the secondary structure elements in the tubulin core. While the tubulin core part shows a resolution ~ 6 Å, the decorated SSNA1 fibril is not resolved due to the symmetry mismatch between microtubules and SSNA1. The surface of the microtubules is not resolved either, presumably due to the coverage of SSNA1 knob-like pattern with 11 nm periodicity, blurring surrounding densities. Note the 11-nm knob-like pattern is averaged out due to the symmetry mismatch to the microtubule symmetry. (E) Chemical crosslinking of microtubules in the absence (top) and presence (bottom) of SSNA1. Subtilisin proteolyzes tubulin E-hooks, and subsequent crosslinking shows the loss of SSNA1-tubulin binding.