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between them. This enabled us to clearly verify that
a
-
a
and
b
-
b
lateral contacts—
and at a lower resolution, the seam
a
-
b
and
b
-
a
lateral contacts—have similar den-
sities. This is despite the divergence of the M and N loop sequences between
a
- and
b
-tubulin.
These native tubulin-tubulin contacts within GDP MTs can now be compared
with other MT reconstructions. We have recently visualized another structural state
of MTs in the absence of stabilizing drugs, but containing the nonhydrolyzable GTP
analog GTP
g
S and bound by another MAP—the fission yeast end binding protein
(EB) Mal3 (
Maurer, Fourniol, Bohner, Moores, & Surrey, 2012
). GTP
g
S acts as a
static mimic of the otherwise dynamic binding site that is specifically recognized
by Mal3 and other EBs at growing MT ends (
Maurer et al., 2011
). The comparison
of this MT end-like structure, with the GDP MT lattice structure in the presence of
DCX, shows that growing MT ends possess an additional layer of lateral contacts at
higher radius, which likely explains their action as a stabilizing cap (see also
Yajima
et al., 2012
). Thus, the comparison of cryo-EM maps of MTs at secondary-structure
resolution is revealing the structural basis of MT dynamic instability.
3.3
DISCUSSION
Cryo-EM has been an essential tool in shedding light on MT stabilization by DCX.
Nevertheless, critical aspects of our current data continue to limit our understanding
of the molecular mechanism of this essential protein. One intriguing aspect of our
reconstructions continues to be that we currently only visualize a single DC-shaped
density in our structures, that is,
1/4 of the FL DCX molecule. This is consistent
with the original low-resolution cryo-EM reconstruction of paclitaxel-stabilized
MTs bound with the t-DCX construct (
Fig. 3.2
;
Moores et al., 2004
), thereby disprov-
ing the hypothesis that our ability to visualize the entire DCX molecule was limited
by the original experimental conditions. In addition, our recent analysis of the struc-
ture of DCX-MTs in the absence of bound kinesin has revealed a specific confor-
mation for the linker regions on either side of the bound DC domain that strongly
suggests the density visualized in all our reconstructions corresponds to N-DC
(
Cierpicki et al., 2006; Liu et al., 2012
).
Recent single-molecule studies have revealed the cooperative nature of MT bind-
ing by DCX, implying that DCX molecules contact each other when present at close
to stoichiometric concentrations on the MT lattice (
Bechstedt & Brouhard, 2012
).
Unfortunately, our structures currently provide no information about these interac-
tions: N-DC—the only domain of DCX we have visualized—is not by itself suffi-
cient either for nucleation, stabilization, or 13-pf specification, for which C-DC
and the C-terminal domain are required (
Kim et al., 2003; Moores et al., 2004;
Sapir et al., 2000; Taylor et al., 2000
). One explanation is that the samples used
for our subnanometer resolution reconstructions are end-points of the DCX-mediated
nucleation and stabilization process. Therefore, some of our current efforts are
directed toward a structural understanding of early species in the process of
