tusc was co - expressed with gst - spc110p as described13,21 , except that complexes were eluted by cleavage of the gst tag with tev protease as the final purification step .
tusc rings were formed by 30 minute incubation on ice after dilution to 0.2 m in brb80 ( 80 mm pipes ph 6.9 , 1 mm egta , 1 mm mgcl2 ) .
the cryo - em reconstruction was performed essentially as described by egelman17 and sasche , et al.25 .
 microtubules are nucleated in vivo by -tubulin complexes . 
 the 300 kda -tubulin small complex ( tusc ) , consisting of two molecules of -tubulin and one copy each of the accessory proteins spc97p and spc98p , is the conserved , essential core of the microtubule nucleating machinery1,2 . in metazoa 
 multiple tuscs assemble with other proteins into -tubulin ring complexes ( turcs ) . 
 the structure of turc suggested that it functions as a microtubule template25 . 
 because each tusc contains two molecules of -tubulin , it was assumed that the turc - specific proteins are required to organize tuscs to match thirteen - fold microtubule symmetry . here 
 , we show that tusc forms rings even in the absence of other turc components . 
 the yeast adaptor protein spc110p stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions . 
 the 8 cryo - em reconstruction of the filament reveals thirteen -tubulins per turn , matching microtubule symmetry , with plus ends exposed for interaction with microtubules , implying that one turn of the filament constitutes a microtubule template . 
 the domain structures of spc97p and spc98p suggest functions for conserved sequence motifs , with implications for the turc - specific proteins . 
 the tusc filaments nucleate microtubules at a low level , and the structure provides a strong hypothesis for how nucleation is regulated , converting this less active form to a potent nucleator . 
Methods Summary
Supplementary Material