Biology Reference
In-Depth Information
viruses. In particular, X-ray crystallography
1
is the only available tech-
nique allowing structure determination at the atomic level, while
recent advances in cryo-EM coupled with developments in image
recognition techniques currently allow modeling of virus struc-
tures up to ~5 Å resolution.
2
Both techniques provide direct three-
dimensional structural information, and they allow visualization of the
interior of the virus as well as its surface. Similarly, transmission elec-
tron microscopy is currently almost the only technique to probe virus-
cell interactions. However, X-ray crystallography and EM have several
distinct limitations. X-ray diffraction requires the virus to be in a crys-
talline form, which is often impossible to achieve. Furthermore, it is
currently limited to the studies of viruses with sizes of up to ~ 50 nm.
It is unlikely that future technological developments will push this
limit beyond 150 nm. Cryo-EM is most useful for highly symmetrical
viruses, such as icosahedral viruses. Both techniques require highly
purified, structurally homogeneous virus samples. Hence, because of
structural heterogeneity, lack of symmetry and large sizes, many ani-
mal and human viruses are not amendable to either X-ray diffraction
or cryo-EM techniques. Another limitation is that both techniques
provide a time-and-space averaged model of the specimen population
and therefore cannot visualize unique features that differ from virion
to virion. Finally, both X-ray diffraction and cryo-EM require sub-
stantial amounts of time for sample preparation, data collection and
calculations.
In the past five to seven years, significant advances have been
made in structural studies of viruses and virus-infected cells by atomic
force microscopy (AFM). The principles of AFM operations have
been described in great detail elsewhere (Dufrene, 2004; Malkin
et al.
,
2005).
3,4
AFM has different virtues that tend to be complimentary to
those of X-ray diffraction and EM. AFM allows rapid visualization of
high-resolution architectures of single virions present in a relatively
crude preparation. This enables access to variations in the particle struc-
ture within a population, which could be important for the evaluation
of their infectivity. AFM provides a resolution comparable to conven-
tional EM methods,
5
while at the same time it can be conducted in
