Structural features, reconstitution and purification of the yeast Ndc80 complex. (a) Schematic of the organization of the Ndc80 complex. It is comprised of two heterodimeric subcomplexes, Nuf2/Ndc80 and Spc24/Spc25. Its elongated, rod-like structure is produced by the coiled-coil regions of each subunit, while capped at both ends by two globular domains. The Nuf2/Ndc80 end interacts with the microtubules while the Spc24/Spc25 end interacts with the centromeric region or other kinetochore components. (b) Coomassie-stained gel showing purification of the Ndc80 complex from SF9 insect cells. MW: Molecular weight marker; WCE: whole cell extract; Ni-NTA: eluate from Nickel-affinity chromatography. (c) Size exclusion chromatography of Ni-NTA eluate from A on a Superdex 200 column. The two peaks in the chromatogram correspond to the full 4-protein Ndc80 complex and to a dimer consisting of Spc24p and Spc25p. Coomassie-stained gel of Superdex 200 fractions in the lower panel demonstrates homogeneity of the reconstituted Ndc80 complex. The fractions used for EM examination are marked.

Visualization of the yeast Ndc80 complex by electron microscopy. (a) Electron micrograph of negatively stained Ndc80 complexes. The scale bar corresponds to 50 nm. (b) Gallery of individual Ndc80 molecules windowed from the micrographs. The designated Head 1, Head 2 and kink are marked on the molecule in panel 1. The scale bar corresponds to 10 nm.

Class averages and docking of the atomic models in Head 1 and Head 2. (a) Five representative class averages of the Head 1. (b) Docking of the atomic model of Ndc80/Nuf2 N-terminal region on the different views represented in the class averages. (c) Five representative class averages of the Head 2. (d) Docking of the atomic model of Spc24/Spc25 C-terminal region on the different views represented in the class averages. In (b) and (d), the threshold of the class averages was adjusted to saturation to show the docking more clearly. The scale bars represent 10 nm.

Distribution of positions and angles of the kink in the particles. (a) Distribution of the position of the kink as measured from Head 1 for all the Ndc80 molecules. The schematic shows the architecture of the Ndc80 molecule with the main structural features under consideration marked. The angle and direction of the kink is defined as illustrated. (b) Distribution of the kink angle as measured for all the molecules. Kinks that appear at angles between 0° (when the particle is straight) and 30° were counted together and evenly distributed within this range, because at these angles, the kink is not as clearly defined (the particle tends to appear curved rather than kinked due to the innate flexibility of the coiled coil).

Alignment of the Ndc80 sequences of several organisms around the region of coiled-coil interruption. An interruption in the coiled-coil region of the Ndc80 protein is predicted across species. This loop region introduces a flexible point within the Ndc80 molecule. Within the loop, secondary-structure prediction indicates the presence of a conserved β-strand segment, suggesting the possibility that the region could be relevant for protein–protein interactions.

The out-of-plane rotations of the five Head 1 models. The five docked Ndc80/Nuf2 atomic models are aligned by in-plane translation, rotation, and flipping within the plane corresponding to the carbon surface on the EM grid. (a) Top views of the aligned models with the carbon plane parallel with the plane of the page. (b) End-on views of the aligned models with the carbon plane perpendicular to the plane of the page.

Model describing the Ndc80 molecule's flexibility at the kink and its Head 1's twist. The elbow-like kink bends within a range of 0° to 120°, and the coiled-coil segment connecting Head 1 and the kink point twists in a constraint range of −90° to 90°.

Model for microtubule kinetochore interaction. (a) Ndc80 complex localized at the kinetochore where its flexibility allows it to interact with incoming microtubules in different orientation but with the right polarity. (b) The microtubule-attached Ndc80 complex still undergoes “flexing” while attached to a spindle microtubule. (c) Possible tension-sensing mechanism mediated by the Ndc80 kinked region: as the kinetochore comes under tension, the Ndc80 complex will only exist in the fully extended conformation.