PDBsum entry 1jff

Structural protein

PDB id: 1jff

Contents

Protein chains

412 a.a. *

426 a.a. *

Ligands

GTP

GDP

TA1

Metals

_ZN

_MG

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Refined structure of alpha beta-Tubulin at 3.5 a resolution.

Authors

J.Löwe, H.Li, K.H.Downing, E.Nogales.

Ref.

J Mol Biol, 2001, 313, 1045-1057. [DOI no: 10.1006/jmbi.2001.5077]

PubMed id

11700061

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.

Figure 4.
Figure 4. Stacking of aromatic residues in the N-terminal b-sheet of a-tubulin. The Figure was generated with O.[45]

Figure 6.
Figure 6. (a) 2F[o] -F[c] density for taxol within the b-tubulin structure shown as C^a trace for clarity. The Figure was generated with O.[45] (b) Stereo view of the taxol site including residues that make direct contact with the taxol molecule. The Figure was generated with Insight II (Biosym Inc.).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 313, 1045-1057) copyright 2001.

Secondary reference #1

Title

Structure of the alpha beta tubulin dimer by electron crystallography.

Authors

E.Nogales, S.G.Wolf, K.H.Downing.

Ref.

Nature, 1998, 391, 199-203. [DOI no: 10.1038/34465]

PubMed id

9428769

Figure 3.
Figure 3 Sequences of pig brain - and -tubulin28 used in the model (in the absence of tubulin sequences from cow we have used its closest known relative). Secondary structure elements are indicated and labelled as for Fig. 4. The tubulin preparations used in our experiments contained a mixture of isotypes. Most of the differences between isotypes are located at the extreme C terminus, which is not visible in our density. In most of the other positions of isotype differences, we arbitrarily chose the residue most similar to the other monomer.

Figure 4.
Figure 4 Ribbon diagram of the tubulin dimer showing -tubulin with bound GTP (top), and -tubulin containing GDP and taxotere (bottom). Labels for strands (in the -subunit) and helices (in the -subunit) are included. The arrow indicates the direction of the protofilament and microtubule axis. a, Stereo front view from the putative outside of the microtubule; b, back view from the putative inside of the microtubule; c, side view. Figures produced with AVS (Advanced Visual; ribbon module from M. Carson and A. Shah). The in-out orientation was determined by reference to reconstructions of intact microtubules9. Such reconstructions show prominent longitudinal ridge on the outside, which in our model would be formed by H11, H12 and the loop between H10 and B9, and shallow inside grooves giving the protofilament a bumpy appearance, corresponding in our model to H1, B3 and the long loops in the N-terminal domain. This represents the most likely arrangement of the dimer, because it buries the nucleotide that is at the non-exchangeable site in (see text). For the nucleotide in to be exchangeable at the plus end of a microtubule, the bottom of the figure would correspond to the plus end. We previously presumed the opposite orientation, based on a comparison of the zinc sheets in negatively stained, stain-glucose, and tannin-glucose embedding, with projection maps of open microtubules of known polarity in negative stain9. Some ambiguity in that determination may be introduced by uncertainty about the exact rotational alignment of the protofilament in the sheets with respect to those in open microtubules and by stain artefacts. The polarity with the plus end down would be consistent with experiments that located the -subunit at the plus end of the microtubule^29 and the -subunit at the minus end^30. Circles in b indicate the positions of Cys 241 and Cys 356, separated by about 8 Å.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd