To determine the molecular-level mechanisms directing tubulin self-association at the early assembly states of tubulin nucleation, we reconstituted in-vitro a minimal model system that mimics the key elements of non-templated cytoskeleton tubulin assembly. Using size-exclusion chromatography coupled with state-of-the-art synchrotron solution X-ray scattering (SEC-SAXS), our cutting-edge home developed analysis tools, in combination with thermodynamic theory of macromolecular self-assembly, and a range of biophysical methods, we determined the active self-organization, structures, and energetics of tubulin proteins and their regulation by fluxes of chemical energy, associated with nucleotide hydrolysis reactions. We focused on conditions that are below the critical conditions for microtubule assembly and determined the interactions between tubulin dimers, the ensemble of structures (tubulin dimers, tubulin oligomers and rings) and their mass fraction, as a function of tubulin concentration. An isodesmic model of tubulin self-association (where dimers are added/removed one at a time) is consistent with our data. Our results provide insight into the self-assembly processes of tubulin, highly relevant also above the critical conditions for microtubule assembly. Read more here.