An in depth analysis of the structure of the NDC 80 protein complex.
The Ndc 80 complex is a rod-shaped protein complex with a globular domain on each end. Through the use of rotary shadowing electron microscopy, the approximate length of the entire Ndc 80 complex was found to be 570 Angstroms. The length of the Ndc 80 complex was then supported by the use of two-color fluorescence light microscopy which stained each end of the Ndc 80 complex different colors. SDS-PAGE revealed that the Ndc 80 complex was comprised of four subunits. The four subunits are called Hec1, Nuf2, Spc24 and Spc25. Atomic structure studies of the four subunits that collectively form the Ndc 80 complex found multiple heptad repeat sequences in all of the subunits. This discovery leads to the hypothesis of a two chain, parallel α-helical coiled coil structure of the rod shaped Ndc 80 complex. This coiled coil hypothesis has since been supported through cryo-electron microscopy as well as helical imaging analysis to show the two chain, parallel α-helical coiled coil structure.
Further calculations and analysis of the four subunits of the Ndc 80 complex suggested that the Hec1 and Nuf2 subunits were both approximately 300-400 Angstroms long, while the Spc24 and Spc25 were approximately 150 Angstroms. Therefore, the combined calculated lengths of the four subunits correlated very well to the experimentally-measured length of the entire Ndc 80 complex . The Hec1 and Nuf2 subunits contribute to one of globular domains as well as the great portion of the coiled coil shaft. The Spc24 and Spc25 subunits complete the shaft and comprise the remaining globular domain. Through the use of two-color immunofluorescence with the 9G3 antibody and polyclonal antibodies, which bind to the N-terminal head of the Hec1/Nuf2 and the C-terminal head of the Spc24/Spc25, it was determined that Hec1/Nuf2 was the microtubule binding site and the Spc24/Spc25 acted as the kinetochore binding site . After the microtubule and kinetochore binding sites were determined, there was a shift in research toward identifying how the actual binding occurs.
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