23 / 44
and the authors suggested that the main limitation on resolu-
tion that we now face may be simply the intrinsic flexibility of
proteins and not the hardware or software that we use. Never-
theless, it is clear that further improvements in microscopes,
detectors, and image processing software will lead to many
more macromolecular complexes that can be solved at resolu-
tions approaching 2.0 A˚ . The growing field of super-resolu-
tion light microscopy has been aimed at surmounting the
fundamental limitation on resolution in light microscopy,
the wavelength of light (~0.5
m
m or 5000 A˚ ). In cryo-EM,
the wavelength of the electrons is typically
<
0.02 A˚ , there-
fore the wavelength of the illumination does not set the phys-
ical limit on resolution.
What biological insight does one gain from higher resolu-
tion? Consider
Fig. 5, which shows a
b
-sheet from the
sheath of a type VI secretion system in
Vibrio cholerae
.
The resolution of this cryo-EM reconstruction
( 21) was
~3.2 A˚ , high enough to allow a complete chain trace of
~600 amino acids in the asymmetric unit of the sheath.
Because the sequences of the two proteins in this asym-
metric unit were known, it was possible to thread this
sequence through the density placing the large and bulky
side chains (as in
Fig. 3) into their corresponding density.
If one had only 5 A˚ resolution, portions of this structure
might have been built correctly but ambiguities would
have existed, such as in the
b
-sheet shown, and it would
be unlikely that the correct connectivity could be estab-
lished. Or consider
Fig. 4 ,where ordered water molecules
are visualized by cryo-EM reconstruction, and suggest that
the resolution is high enough to understand enzymatic reac-
tions or design drugs.
CONCLUSIONS
The rapid advances in cryo-EM over the past several years
make it nearly impossible to predict where the field will
be in several years. It is reasonable to expect that the expo-
nential growth of near-atomic resolution structures deter-
mined by cryo-EM
( Fig. 1) will continue, but we still do
not know possible limits in resolution or how large a com-
plex must be to reach near-atomic resolution. The future
of cryo-EM is certain to be exciting, with new biological in-
sights gained from this powerful technique.
REFERENCES
1.
Watson, J. D., and F. H. Crick. 1953. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 171:737–738 .FIGURE 4 A small region from the 2.2 A˚ resolu-
tion cryo-EM reconstruction of
b
-galactosidase
( 20). The resolution is high enough to see an
ordered and bound water molecule in the center
(
yellow
). To see this figure in color, go online.
FIGURE 5 A region from the type VI secretion system sheath of
Vibrio
cholerae
( 21), reconstructed by cryo-EM at ~3.2 A˚ resolution. The atomic
model that was built into the reconstruction (
transparent gray surface
) has
three different molecules shown in cyan, red, and blue. At a resolution worse
than ~4 A˚ , one might have ambiguities in tracing the individual polypeptide
chains present in each molecule. The resolution that was achieved prevented
any such ambiguities, and it was clear that the
b
-sheet shown involved two
strands from one molecule (
cyan
), and one strand from each of two different
molecules (
red
and
blue
). To see this figure in color, go online.
Biophysical Journal 110(5) 1008–1012
The Current Revolution in Cryo-EM
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