Biology Reference
In-Depth Information
3.2.3
Cryo-EM and subnanometer resolution 3D structure
determination
3.2.3.1
Introduction
Using cryo-ET, we demonstrated that DCX promoted the assembly of B-lattice 13-pf
MTs. These MTs contain a discontinuity—the seam—that means that they are not
amenable to helical reconstruction. Instead 13-pf MTs can be reconstructed using
so-called single-particle image processing. The method developed by Charles Sin-
delar (Yale, CT;
Sindelar & Downing, 2007, 2010
), referred to as “Chuff,” yielded
an
8
˚
resolution reconstruction of kinesin-decorated, DCX-stabilized 13-pf MTs,
the same sample that we had validated by cryo-ET (
Section 2.2
) (referred to below as
DCX-K-MTs;
Fourniol et al., 2010
).
3.2.3.2
Sample preparation and data collection
Sample preparation was as described in
Section 2.2.2
. Low-dose images were col-
lected on a Tecnai F20 FEG electron microscope (FEI Company), operating at
200 kV, 50,000
, and 0.8-2.9
m
m defocus. Micrographs were recorded on film
(SO-163; Kodak) and were digitized (SCAI scanner; Carl Zeiss, Inc.) to a final sam-
pling of 1.4
˚
/pixel.
3.2.3.3
Setup and automated processing
Chuff is a fully automated set of scripts that simply require the user to set up the data
and parameter files. To speed up image processing, and because finer sampling did
not significantly improve resolution, micrographs digitized to 1.4
˚
/pixel were binned
by a factor of 2 to a final sampling of 2.8
˚
/pixel. For our study, DCX-K-MTs were
boxed with the EMAN Boxer program (option helix/normal;
Ludtke, Baldwin, &
Chiu, 1999
) into particles/segments of 432
432 pixels, containing 7-8 tubulin dimer
axial repeats. The input for the DCX-K-MT reconstruction was 4772 segments
coming from 172 MTs and 63 cryo-electron micrographs.
3.2.3.4
Initial 3D model and reference projections
We used the default initial 3D model of kinesin-decorated 13-pf B-lattice MT gen-
erated by Chuff for alignment. In particular, the kinesin head density bound every
dimer greatly facilitates identification of the position of the MT seam. The absence
of DCX in the initial model gave us confidence that additional density observed in
the output 3D reconstruction is indeed a faithful reflection of the MT-bound DCX in
our sample. To further minimize the risk of model bias, high-resolution information
was deleted using a low-pass filter with a 15
˚
cut-off. Two-dimensional projections
of the model with
varying between 0
and 359
,
between 75
and 105
, both with
y
'
a1
increment, resulted in a set of 11,160 references.
3.2.3.5
Finding the seam and image selection
In Chuff, a first round of reference-based alignment is performed in SPIDER (
Frank
et al., 1996
), whereMT segments are cross-correlated to the references. Each segment
cut along one MT image is assigned a seam orientation (
angle). Because the seam
'
