Fig. 2: Time-dependent synchrotron SAXS data with increasing divalent cations in PIPES buffer at pH 6.8 reveals an intermediate bundled (B_int) microtubule state between the bundled wide-spacing (B_ws) and the tubulin ring states.

A SAXS data (open circles) and corresponding fits (solid lines) for increased Ca²⁺ concentrations at t₀, where t₀ is the time of the first measurement. Color of the fitted line represents the identified tubulin phase with green, yellow, and magenta, corresponding to profiles in the wide-spacing bundled (B_ws), intermediate bundled (B_int), and depolymerized tubulin ring states respectively. Indexing of two lower profiles is for a 2D hexagonal lattice of MTs. The B_int (profiles at 1.5 mM and 1.6 mM Ca²⁺) is differentiated from the B_ws (profile at 1.2 mM Ca²⁺) by peaks shifted to larger q (i.e. smaller lattice spacing), decreased peak widths (compare 1,0 peak widths for 1.6 mM and 1.2 mM Ca²⁺), and increased scattering intensity at local minima of the MT form factor (solid arrows at 1.2 mM, 1.5 mM, and 1.6 mM Ca²⁺). At 2.5 mM of added Ca²⁺, the SAXS is dominated by tubulin rings and curved oligomers. B Cartoon of hexagonal MT bundles in the B_ws (top) and B_int, (bottom) states highlights changes in MT-MT spacing. C Time-dependent SAXS data (open circles) and corresponding fits (solid lines) of a sample with 1.2 mM added Ca²⁺. Evolution of SAXS profiles show that phase transitions occur from B_ws to B_int (between t₀ + 12 and t₀ + 18 hrs) and from B_int to the tubulin ring state (between t₀ + 18 and t₀ + 30 hrs). Source data for A and C are provided in the Source Data file.