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gel filtration was performed last but the modified order improved yields by reducing
degradation and aggregation (see
Fig. 3.3
Cii). The His-tag may make the protein
more soluble, while performing the gel filtration at an early stage helps eliminate
contaminants including proteases that might cause both instability and degradation.
Finally, the
10 mL dilute flow-through is concentrated in a 10,000-molecular-
weight cut-off concentrator (Vivaspin 15R; Sartorius). Centrifugation at 4000
g
,
always at 4
C, for 1 h concentrates DCX approximately 5
2 mL at 5 mg/mL).
The concentrated protein is aliquoted and snap-frozen in liquid nitrogen and keeps
for several years at
(
80
C.
3.2.2
Cryo-ET and evaluation of DCX-MT architecture
3.2.2.1
Introduction
In the different MT architectures polymerized
in vitro
, lateral contacts between pfs
are usually homotypic—
a
-
a
and
b
-
b
—forming a so-called B-lattice. However, het-
erotypic contacts—
a
-
b
and
b
-
a
, the so-called A-lattice—also occur (
Wade &
Chr
´
tien, 1993
). Although the precise
in vivo
significance for MT architecture is
far from clear, crucially for our studies, 13-pf MTs show a line of discontinuity called
the seam, where the dominant B-lattice contacts are disrupted by a line of A-lattice
contacts (
Kikkawa, Ishikawa, Nakata, Wakabayashi, & Hirokawa, 1994
). However,
13-pf MTs built entirely of A-lattice contacts have also been reported (
des Georges
et al., 2008
).
Subnanometer resolution structure determination by single-particle processing
requires an initial 3D reference model, which implies some prior knowledge of
the structure of interest. In our case, it was well established that copolymerized
DCX-MTs are 13-pf, but it was unclear whether they were made of mainly A- or
B-lattice contacts or a mixture. Although previous reports showed that truncated
DCX bound to paclitaxel-stabilized MTs that are built of a helical B-lattice
(
Moores et al., 2004
), we considered the hypothesis that DCX-MTs might contain
a proportion of A-lattice contacts. Cryo-ET permits 3D reconstruction from a single
macromolecular complex without averaging or model bias and is thus ideally suited
to determine the lattice parameters of a population of MTs.
McIntosh et al. (2009)
successfully applied cryo-ET to MTs extracted from cells and decorated with a kine-
sin motor domain, and observed primarily B-lattice MTs. We took a similar ap-
proach, using K340
T93N
, a 340-residue Thr93Asn mutant motor domain of rat
conventional kinesin to emphasize the underlying lattice of our DCX-MTs
(
Crevel et al., 2004
). This mutant has a very strong affinity for MTs facilitating full
decoration but did not alter the stoichiometry of DCX binding (validated by cosedi-
mentation assay, data not shown).
3.2.2.2
Sample preparation
DCX-MTs were polymerized by incubating bovine tubulin (Cytoskeleton, Inc.) and
human recombinant DCX in equimolar amounts (10
m
M) at 37
C for 1 h. To maxi-
mize decoration of MTs with DCX and kinesin, DCX-MTs were diluted 1:1 in the
