Detailed Structure of Single, Unaveraged Microtubules Revealed by Electron Microscopy Tomography

Abstract

Tubulin carboxyterminal tail peptides are short (10-20 amino acids) acidic, unstructured peptides that vary significantly in sequence between tubulin isotypes and are exposed on the outer surface of microtubules. These peptides have, so far, eluded direct visualization. Electron microscopy tomography is used here to analyze stabilized microtubules embedded in a tungsten-based negative stain previously found to resist sustained electron beam irradiation. Sample stability is compatible with high electron doses, permitting collection of 142 images at various tilt angles with high signal-to-noise ratio. The resulting tomogram is reconstructed without the need of averaging multiple samples. After further optimizing collection of data and signal elaboration, images reveal the three dimensional structure of subunits as well as previously unresolved small stalks on the outer surface of the microtubule. Inspection of virtual sections demonstrates that the stalks are ∼ 2.5 nm long and ∼ 1 nm wide (at half length). Stalks protrude intermittently every ∼ 4 nm along the microtubule from the outer face of each monomer. These protrusions are well resolved from background in virtual sections perpendicular to the long axis of microtubule. These protrusions are likely the carboxyterminal peptides of tubulin. This hypothesis is supported by comparison with results obtained in the same way from microtubules whose C-terminals have been cleaved enzymatically. In summary, it is possible to resolve single peptides by amplitude contrast in a conventional electron microscope without the need of averaging data from multiple microtubules.