Fig. 6: Plastic-embedded TEM confirms the existence of the B_ws and B_int bundled phases.

Electron microscopy of microtubule assemblies prepared at 37 °C with mixtures of tau, tubulin, and 2 mM GTP in PIPES buffer at pH 6.8 with 1.8 mM added Mg²⁺ and fixed after 3 h (A–C) and 18 h (D–F). Time points for sample fixation were selected based on SAXS data for samples prepared with identical conditions, where the wide-spacing B_ws and the intermediate B_int bundled phases were present at 3 and 18 h, respectively. A, D Top panels depicting cross-sections at low magnification show the extent of MT bundling for both phases (B_ws state in A and B_int state in D). The images further show the propensity for MT bundles to arrange in linear arrays (dashed white boxes). B, E Higher magnification cross-sections at 3-hour and 18-hour timepoints highlight the larger MT-MT spacing in the B_ws compared to B_int phase and the larger number of MT-MT cross-bridges per MT (and larger average number of neighbors for each MT) in the B_int compared to the B_ws phase. C, F Low magnification side views at 3-hour and 18-hour timepoints show that the width of the MT bundles (i.e. bundle size) is much larger in the B_int compared to the B_ws phase, and the spacing between MTs is smaller in the B_int compared to the B_ws phase, consistent with trends observed in SAXS data. All samples contained the stated Mg²⁺ concentrations added to standard PIPES buffer at pH 6.8, which includes 1 mM of Mg²⁺ (see Methods). Raw TEM images are provided in the Source data file.