Biology Reference
In-Depth Information
and Chiu 2003).
35,36,39
If high-resolution crystallographic models of
the individual proteins of a macromolecular complex are available (as
is the case for the HSV-1 capsid structures), these atomic-resolution
models can be fitted into the corresponding portions of the cryoEM
map using the identified secondary structure elements as internal
markers to guide the fitting, thereby establishing a pseudo-atomic
model of the entire assembly. The large size of the herpesviruses
(over 200 nm) and the presence of partially ordered tegument pro-
teins inside the virion make cryoEM the method of choice for deter-
mining the structures of herpesvirus capsids and related particles.
The use of higher-voltage cryo-electron microscopes — which offer
greater penetrating power for reducing inelastically scattered elec-
trons and increased depth of focus for imaging thicker specimens —
is particularly important for imaging herpesvirus particles, which are
among the largest animal virus studies to date. Improved data pro-
cessing software, such as the IMIRS
40,41
and PFT packages
42
and oth-
ers, also facilitates the automation and management of the large
amount of data required to compute higher-resolution structures.
In recent years, cryoET has gained popularity as a tool for exam-
ining the 3D structures of macromolecular complexes that are too
large, asymmetrical, or heterogeneous to be investigated by conven-
tional cryoEM or crystallographic methods (reviewed by Subramaniam
and Milne).
43
Rather than averaging the structures of many particles,
as is the case in cryoEM, cryoET can be used to reconstruct a single,
asymmetric particle without averaging.
44-48
Projection images of the
ice-embedded sample are taken over a range of tilt angles. The 2D
information from these images is merged to obtain 3D tomograms,
which can be examined as a series of thin, serial slices. Specific molecules
can be identified in cryoET reconstructions, indicating the potential
for cryoET to approach true molecular resolution.
46,49
Recent advance-
ments in high pressure freezing and cyro-sectioning
46,50,51
show great
promise for studying intact, virus-infected cells using serial sections
that are thin enough for tomographic studies. Enveloped viruses, such
as herpesviruses, and their infection processes, can be visualized in
3D through tomographic reconstructions.
