Research

-Tubulin Small Complex (-TuSC) and -Tubulin Ring Complex (-TuRC) are microtubule nucleating (MT) complexes in the microtubule organizing centers (MTOCs). -TuSC is a highly conserved 300kDa heterotetramer and forms part of the 2.2MDa -TuRC in metazoa. The overall architecture of the -TuSC in yeast (Tub4 complex) and -TuRC in Drosophila has been elucidated by Electron Microscopy (EM). One study showed conformation variability on the Tub4 complex and it was proposed as a key point in regulation, while the other showed that the architecture of -TuRC was consistent with the template model for MT nucleation. Despite this insights, we still need to understand how these complexes achieve and regulate MT nucleation.In addition to the biological information, the studies on -TuRC and -TuSC revealed that both complexes have conformational heterogeneity; this poses a problem in obtaining high-resolution 3D structures in EM due to potential classification of the data into many classes and incoherence due to misclassification and misalignment. My project addresses the understanding of -Tubulin complexes in Drosophila where no structure of the -TuSC is known and there is very little information on the -TuRC structure. To do so, I will use conventional EM techniques to elucidate features of the structures, but to address the goal of obtaining a high resolution structure of -TuRC, I am going to try to develop a method to facilitate obtaining high resolution structures of asymmetrical complexes with conformational heterogeneity.

My aims are:

Aim 1: Preliminary structure and structural characterization of Drosophila -TuSC and -TuRC.

Aim 2: A new single particle approach to improve resolution of 3D reconstructions of macromolecules with conformational heterogeneity.

Aim 3: Applying Aim 2 in cryo-EM, while obtaining a higher resolution structure of -TuRC.

Publications

Choy RM, Kollman JM, Zelter A, Davis TN, Agard DA, "Localization and orientation of the gamma-Tubulin Small Complex components using protein tags as labels for single particle EM, J Struct Biol. 2009 Aug 29. (html, pdf)