Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 313, Issue 5, Pages 1045-1057Publisher
ACADEMIC PRESS LTD
DOI: 10.1006/jmbi.2001.5077
Keywords
tubulin; taxol; zinc-sheets; electron crystallography; crystallographic refinement
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Funding
- NIGMS NIH HHS [GM46033] Funding Source: Medline
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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.
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