Biology Reference
In-Depth Information
FIGURE 3.2
Low-resolution cryo-EM reconstruction reveals doublecortin's unique MT-binding site
between protofilaments. (A) Front view of the 3D difference map showing density attributable
to t-DCX (yellow), wedged between the pfs and over one of the two distinct fenestrations
of the undecorated MT 3D map (blue). (B) View from the MT plus-end, illustrating how DCX
fits into the inter-pf valley. The asterisk marks the paclitaxel-binding site. The green
and pink densities show, respectively, kinesin- and MAP2/tau-binding sites on one pf. Scale
bars
20
˚
(
Moores et al., 2004
).
¼
dimers. We also wanted to understand how DCX would stabilize MTs in the absence
of the stabilizing drug paclitaxel. Therefore, we have focused our efforts on subnan-
ometer resolution structural studies of 13-pf MTs nucleated and stabilized by
FL DCX.
3.2
METHODS
3.2.1
Expression and purification of recombinant human DCX
3.2.1.1
Introduction
Early biochemical studies used recombinant DCX expressed in
E. coli
(
Sapir et al.,
2000; Taylor et al., 2000
). However, when we started our work on DCX, our
bacterially expressed protein produced primarily large 3D bundles of MTs that were
impossible to study structurally, even at low-protein concentrations. On the other
