Electron microscopy of metal-shadowed and negatively stained microtubule protein. Structure of the 30 S oligomer.

Microtubule protein purified from porcine brain was fixed at low protein concentration with glutaraldehyde under conditions which maximize the relative concentration of the ring-shaped 30 S oligomer. Fixed oligomer was separated from glutaraldehyde and other protein species by column chromatography. The fixed, isolated oligomer was deposited on electron microscopy grids, dehydrated, and then critical point-dried before shadow-coating with carbon/platinum alloy at a fixed angle. Analysis of the shadow lengths observed by electron microscopy revealed that the height of the 30 S oligomer is 15 nm. Microtubule protein deposited on electron microscope grids at high protein concentrations was examined by the negative stain technique and found to contain apparent stacks of oligomer from which the number of tubulin dimers per turn of the ring and the distance between turns could be determined. The number of subunits per turn was determined as 13.8. The distance between turns was found to be 7.4 nm, indicating that the 15 nm high, shadowed oligomers consisted of two turns. Additional information from the literature is considered and a model is presented for the oligomer. The model is a helix of 29 tubulin dimers and five high molecular weight protein molecules arranged so as to preserve intersubunit bonding patterns found in microtubules.