Reconstructions of tubulin protofilaments: different appearances of the same structure

We compare the structures of tubulin protofilaments obtained by image reconstruction of tubulin sheets and hoops, and by X-ray diffraction of microtubules. Negatively stained specimens yield up to eight different appearances of protofilaments. They all represent the same intrinsic structure. The effects are explained by the packing of the protein subunits and the stain distribution resulting from it. They cannot be interpreted directly in terms of protein structure or composition; in particular, differences in staining cannot be attributed to microtube-associated proteins (MAPs). None of the reconstructions reproduce the X-ray structure faithfully. The discrepancies between the reconstructions are seen at resolutions of 4 nm or less. This appears to be the limit of structural fidelity of negatively stained tubulin specimens, even when the nominal resolution of the micrographs is better. Most of the differences may be defined in terms of the diffraction patterns and are therefore genuine. Additional differences become apparent after computing the 3D reconstruction and may be genuine and/or due to the data treatment. Several factors affecting the appearances are discussed.