This application is concerned with the reconstitution of the outer kinetochore, and its analysis by biophysical techniques and electron microscopy. The kinetochore is a large multi-subunit and multi-copy protein complex that bridges mitotic chromosomes with spindle microtubules. The kinetochore-microtubule interface is crucially important for faithful division of replicated DNA during mitosis and is therefore subtly regulated to perform its functions optimally, maintaining genome stability. Failures in the chromosome segregation process are associated with genetic diseases and cell transformation.To understand the structural organization of the kinetochore-microtubule interface, a clear overview of the relevant players and their role and position in the kinetochore is needed. The binding of kinetochores to microtubules likely involves cooperative interactions between different copies of the kinetochore components. Several kinetochore proteins are known to bind microtubules, but the main mediator of the ‘end-on’ microtubule interaction is the NDC80 complex. Previous structural work from our laboratory has shed light on the organization of a minimal NDC80 complex and its mechanism of microtubule binding. Building on these previous studies, it is now important to extend the analysis to the function of the entire kinetochore.To study the binding of the kinetochore to microtubules in more detail, we now propose to reconstitute the full-length NDC80 complex with additional recombinantly expressed kinetochore components, including CENP-C, the MIS12 complex, the SKA complex and the KNL1 complex. The influence of these additional components on microtubule binding will be studied using biophysical approaches and electron microscopy. Through this we will gain insight into 1) the structural basis of kinetochore organization, 2) the conformational changes upon microtubule binding, and 3) the dynamics of relevant interactions.