To shed light onto the activation mechanism of vinculin, we carried out a detailed refinement of chicken vinculin and compared it to the human protein which is greater than 95% identical. Refinement resulted in a complete and significantly improved model. This model includes important elements such as a pro-rich strap region (PRR) and C-terminus. The conformation of the PRR stabilized by its inter- and intra-molecular contacts shows a dynamic, but relatively stable motif that constitutes a docking platform for multiple molecules. The contact of the C-terminus with the PRR suggests that phosphorylation of Tyr1065 might control activation and membrane binding. Improved electron densities showed the presence of large solvent molecules such as phosphates/sulfates and a head-group of PIP2. The improved model allowed for a computational stability analysis to be performed by the program Corex/Best which located numerous hot-spots of increased and decreased stability. Proximity of the identified binding sites for regulatory partners involved in inducing or suppressing the activation of vinculin to the unstable elements sheds new light onto the activation pathway and differential activation. This stability analysis suggests that the activation pathway proceeds by unfurling of the super-bundle built from four bundles of helices without separation of the Vt region (840-1066) from the head. According to our mechanism, when activating proteins bind at the strap region a separation of N and C terminal bundles occurs, followed by unfurling of the super-bundle and flattening of the general shape of the molecule, which exposes the interaction sites for binding of auxiliary proteins.