Refined structure of αβ-tubulin at 3.5 A resolution

被引:974
作者
Löwe, J
Li, H
Downing, KH
Nogales, E [1 ]
机构
[1] Lawrence Berkeley Natl Lab, Div Life Sci, Berkeley, CA 94720 USA
[2] MRC, Mol Biol Lab, Cambridge CB2 2QH, England
[3] Univ Calif Berkeley, Howard Hughes Med Inst, Berkeley, CA 94720 USA
关键词
tubulin; taxol; zinc-sheets; electron crystallography; crystallographic refinement;
D O I
10.1006/jmbi.2001.5077
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We present a refined model of the ap-tubulin dimer to 3.5 Angstrom 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. (C) 2001 Academic Press.
引用
收藏
页码:1045 / 1057
页数:13
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