Refined structure of αβ-tubulin at 3.5 Å resolution 1 1Edited by I. A. Wilson

Abstract

We present a refined model of the alpha beta-tubulin dimer to 3.5 A resolution. An improved experimental density for the zinc-induced tubulin sheets was obtained by adding 114 electron diffraction patterns at 40-60 degrees tilt and increasing the completeness of structure factor amplitudes to 84.7 %. The refined structure was obtained using maximum-likelihood including phase information from experimental images, and simulated annealing Cartesian refinement to an R-factor of 23.2 and free R-factor of 29.7. The current model includes residues alpha:2-34, alpha:61-439, beta:2-437, one molecule of GTP, one of GDP, and one of taxol, as well as one magnesium ion at the non-exchangeable nucleotide site, and one putative zinc ion near the M-loop in the alpha-tubulin subunit. The acidic C-terminal tails could not be traced accurately, neither could the N-terminal loop including residues 35-60 in the alpha-subunit. There are no major changes in the overall fold of tubulin with respect to the previous structure, testifying to the quality of the initial experimental phases. The overall geometry of the model is, however, greatly improved, and the position of side-chains, especially those of exposed polar/charged groups, is much better defined. Three short protein sequence frame shifts were detected with respect to the non-refined structure. In light of the new model we discuss details of the tubulin structure such as nucleotide and taxol binding sites, lateral contacts in zinc-sheets, and the significance of the location of highly conserved residues.

Evolution of the cytoskeleton

“…In order to further study the evolution of tubulins from FtsZ, I have prepared a new sequence alignment, relying on recent structure-based pairwise alignments, (34)(35)(36) and have identified amino acids that are highly conserved in FtsZ and in α, β and γ tubulins. This was originally done by Mukherjee and Lutkenhaus,(37) and the resulting sequence identities, although limited, provided the strongest early argument for homology of FtsZ and tubulin.…”

A driving and coupling “Pac-Man” mechanism for chromosome poleward translocation in anaphase A

“…The protofilament is a series of N GDP-tubulin subunits connected end-to-end (N ϭ 500 in the calculation corresponding to 4 m length). Each subunit is modeled as a rigid rod of 8 nm long (l ϭ 8 nm) (24), carrying a highly negative charge Q MT Ϸ 50 e per subunit (24,25). The bound GDP-tubulin subunit has a preferred angle with respect to its neighbor (0) ϭ 0.4 rad (24).…”

Immunogold method evidences that kinesin and myosin bind to and couple microtubules and actin filaments in lipotubuloids of Ornithogalum umbellatum ovary epidermis

“…Flexibility of microtubules connected with the fact that microtubule subunits are bound more strongly inter-protofilaments than within-protofilaments in vitro was shown by many authors (Chrétien et al 1998;Nogales 1999;Nogales et al 1999;Löwe et al 2001;Meurer-Grob et al 2001;Kis et al 2002;Sept et al 2003;Krebs et al 2005;Tuszyński et al 2005;Wang and Nogales 2005;Nogales and Wang 2006;Drabik et al 2007;Hunyadi and Jánosi 2007).…”