“Cryo-EM Structure of the g-Tubulin Small Complex”
Justin Kollman – Department of Biochemistry and Biophysics, University of CA San Francisco
Microtubules are nucleated in vivo by complexes containing g -tubulin. The core of the microtubule nucleating machinery, conserved in all eukaryotes, is the g -tubulin small complex (g -TuSC), a 300 kDa heterotetramer containing two copies of g -tubulin. In most eukaryotes multiple g -TuSCs assemble with a number of other proteins into large g -tubulin ring complexes (g -TuRCs); budding yeast, however, have only g -TuSC and lack the other g -TuRC-specific components. We have therefore been studying yeast g -TuSC as the minimal nucleating complex. Previously, we determined the structure of monomeric yeast g -TuSC in negative stain by the random conical tilt (RCT) method. Here we show that g -TuSC forms helical filaments when in complex with the N-terminal domain of Spc110, which binds g -TuSC to the nuclear face of the spindle pole body. We have determined the cryo-EM structure of these filaments by iterative helical real space reconstruction (IHRSR). g -TuSCs associate laterally in the helix, presenting the g -tubulins in an arrangement nearly identical to that of tubulin in the microtubule, supporting previous models of nucleation which suggested that g -tubulin forms a template for microtubule growth. The helix has 6.5 g -TuSCs, or 13 g -tubulins, per turn, matching the predominant 13 protofilament organization of microtubules in vivo. We propose that a single turn of this helix is equivalent to the core of g -TuRC.