Tau is a microtubule-associated protein that plays a critical role in regulating the organization and stability of microtubules (MTs) in neurons. Although the association of tau with MTs is well recognized, the structural consequences of its binding at steady state have remained poorly understood. Here, we combined small-angle x-ray scattering with AlphaFold predictions and high-resolution modeling using our D+ software to investigate how tau modulates the MT architecture in-vitro. We reconstituted a minimal model system of dynamic MTs with purified tubulin and then added either full-length tau (FL-tau, or 2N4R) or a minimal four-repeats tau construct (4R-tau). We applied two assembly protocols: addition of tau to preassembled MTs and coassembly of tau with tubulin. Our analysis revealed that the two assembly protocols resulted in very similar structures at steady state. Tau altered the MT lattice organization in a construct-dependent manner. Specifically, tau binding increased the mass fraction of tubulin dimers in MTs with 15 protofilaments, whereas the largest mass fraction of tubulin still formed 14-protofilament MTs. Tau also limited the number of tubulin dimers in positional correlation along the longitudinal MT-axis. This result suggests that tau promoted tubulin nucleation and stabilized shorter MTs. These effects were more significant with FL-tau than with 4R-tau. In addition, the critical tubulin concentration for MT assembly decreased by about 20% in the presence of tau, and tau formed larger (non-MT) small tubulin complexes that coexisted with MT or tau-coated MT. Our results show that tau modified the lateral and longitudinal tubulin interactions and modulated the MT architecture in a manner that is likely to be relevant to its function in-vivo.
