Native yeast kinetochore structures identify an essential inner kinetochore interaction

Kinetochores must accurately assemble on centromeres every cell cycle for faithful chromosome segregation. Although a conserved centromeric histone variant is essential for inner kinetochore formation, the budding yeast centromeric DNA is a poor template for nucleosome formation in vitro, possibly due to a resistance to bend. To better understand how the yeast inner kinetochore is assembled, we developed a one-step protocol to purify de novo assembled native inner kinetochore subcomplexes for structural studies. We performed cryoelectron microscopy on the purified complexes and generated medium to high resolution density maps of four separate inner kinetochore complexes, two of which had not previously been visualized. We detected differences between native and previously reconstituted structures, suggesting that the de novo assembly assay generated intermediate assemblage states. A strong extra structural density, which corresponds to an Ndc10 trimerization domain, associated with centromeric DNA and a pair of CBF3 complexes to induce significant centromere bending. Its deposition on the CBF3-CEN complex is essential for kinetochore assembly and chromosome segregation. We propose that Ndc10 trimerization facilitates bending of the centromeric DNA, leading to assembly and stabilization of the centromeric nucleosome and inner kinetochore.