Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE
561
NUCLEUS
CYTOSOL
nuclear
pore
Figure 9–52 The nuclear pore. Rapidly
frozen nuclear envelopes were imaged in
a high-resolution SEM, equipped with a
field emission gun as the electron source.
These views of each side of a nuclear pore
represent the limit of resolution of the SEM
(compare with Figure 12–8). (Courtesy of
Martin Goldberg and Terry Allen.)
50 nm
being plunged into a coolant. A special sample holder keeps this hydrated specimen
at –160°C in the vacuum of the microscope, where it can be viewed directly
without fixation, staining, or drying. Unlike negative staining, in which what we
see is the envelope of stain exclusion around the particle, hydrated cryoelectron
microscopy produces an image from the macromolecular structure itself. However,
the contrast in this image is very low, and to extract the maximum amount
of structural information, special image-processing MBoC6 m9.51/9.52 techniques must be used, as
we describe next.
Multiple Images Can Be Combined to Increase Resolution
As we saw earlier (p. 532), noise is important in light microscopy at low light levels,
but it is a particularly severe problem for electron microscopy of unstained macromolecules.
A protein molecule can tolerate a dose of only a few tens of electrons
per square nanometer without damage, and this dose is orders of magnitude
below what is needed to define an image at atomic resolution.
The solution is to obtain images of many identical molecules—perhaps tens
of thousands of individual images—and combine them to produce an averaged
image, revealing structural details that are hidden by the noise in the original
images. This procedure is called single-particle reconstruction. Before combining
all the individual images, however, they must be aligned with each other.
Sometimes it is possible to induce proteins and complexes to form crystalline
arrays, in which each molecule is held in the same orientation in a regular lattice.
In this case, the alignment problem is easily solved, and several protein structures
have been determined at atomic resolution by this type of electron crystallography.
In principle, however, crystalline arrays are not absolutely required. With the
help of a computer, the digital images of randomly distributed and unaligned molecules
can be processed and combined to yield high-resolution reconstructions
(see Movie 13.1). Although structures that have some intrinsic symmetry make
the task of alignment easier and more accurate, this technique has also been used
for objects like ribosomes, with no symmetry. Figure 9–54 shows the structure of
What we don’t know
• We know in detail about many cell
processes, such as DNA replication
and transcription and RNA translation,
but will we ever be able to visualize
such rapid molecular processes in
action in cells?
• Will we ever be able to image
intracellular structures at the resolution
of the electron microscope in living
cells?
• How can we improve crystallization
and single-particle cryoelectron
microscopy techniques to obtain highresolution
structures of all important
membrane channels and transporters?
What new concepts might these
structures reveal?
100 nm
Figure 9–53 Negatively stained actin
filaments. In this transmission electron
micrograph, each filament is about 8 nm in
diameter and is seen, on close inspection,
to be composed of a helical chain of
globular actin molecules. (Courtesy of
Roger Craig.)