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runs parallel to the axis of the MT, one would expect that the
values assigned to
segments from the same 13-pf MT would be very similar. Typically, however, they
vary by multiples of
'
28
(360/13) because cross-correlation scores computed be-
tween references whose seam is rotated by an integer numbers of pfs and relatively
noisy cryo-EM images are very similar; hence, the best score does not always corre-
spond to the actual orientation of the seam in the image (
Sindelar &Downing, 2007
).
Alternatively, variations of the
angle can arise from MTs with different architec-
tures, and Chuff contains AWK scripts that analyze the output
'
angles from
reference-based alignment and decide whether each MT has consistent enough seam
orientations (
'
values) to be kept for the reconstruction. This decision depends on a
number of parameters that can be defined by the user, in particular, the size of the an-
gular windowwhere
'
values can be considered to be consistent between one another
(default: 20
), and the minimum fraction of the boxes that must be in this window (de-
fault: 20%). The default selection parameterswere successfully used to process DCX-
K-MTs and approximately 90%of the dataset passed this selection process. If theMT
is accepted, the angles assigned are edited so that all segments in theMT have
'
values
within the previously determined angular window. Subsequent rounds of reference-
based alignment are restricted to this
'
window and performed to refine alignment
parameters and Euler angles before the final 3D reconstruction.
'
3.2.3.6
Reconstructions of DCX-K-MTs
Reconstruction of DCX-K-MTs with no symmetry imposed yielded a 13.5
˚
reso-
lution 3D map (FREALIGN option helical_subunits
0
Grigorieff, 2007
);
Fig. 3.5
A; EMDB ID 1787). This asymmetric reconstruction confirmed that the
MT (cyan) is occupied by both kinesin motor domain (red) and DCX (yellow), which
binds at the corner of four tubulin dimers, stabilizing both lateral and longitudinal
tubulin-tubulin contacts. Remarkably, DCX does not bind at the A-lattice seam
of the 13-pf MT (right panel, arrow), but only at the 12 B-lattice inter-pf grooves
(
Fourniol et al., 2010
).
To gain further insight, the 12 inter-pf valleys bound by DCX were averaged
together. The averaging of approximately 168,000 decorated tubulin dimers gener-
ated an 8.2-
˚
¼
12;
Fig. 3.5
B;
EMDB ID 1788). At this resolution, secondary structures are resolved: alpha helices
and beta sheets appear, respectively, as rods and sheets of density.
resolution map (FREALIGN option helical_subunits
¼
3.2.3.7
Pseudo-atomic model building
The detail in our 8.2
˚
structure of DCX-K-MTs allowed us to dock atomic structures
of each constituent subunit into our reconstruction in order to generate a pseudo-
atomic model of DCX-stabilizedMTs. Such modeling provides invaluable data about
the binding interfaces between the components and the conformational changes they
undergo in the context of the physiologically relevant macromolecular complex. We
focus here on the interactions between DCX and tubulin; the implications of kinesin
binding on DCX have recently been discussed elsewhere (
Liu et al., 2012
).
