Essential Cell Biology 5th edition

169
CRYOELECTRON MICROSCOPY
X-ray crystallography remains the first port of call
when determining proteins’ structures. However, large
macromolecular machines are often hard to crystallize,
as are many integral membrane proteins, and for
dynamic proteins and assemblies it is hard to access
different conformations through crystallography
alone. To get around these problems, investigators are
increasingly turning to cryoelectron microscopy
(cryo-EM) to solve macromolecular structures.
molecules immobilized in thin film of ice
carbon film on EM grid
In this technique, a droplet of the pure protein in water is placed
on a small EM grid that is plunged into a vat of liquid ethane at
−180ºC. This freezes the proteins in a thin film of ice and the rapid
freezing ensures that the surrounding water molecules have no
time to form ice crystals, which would damage the protein’s shape.
beam of electrons
electron detector captures projected image of molecules
The sample is examined, still frozen, by transmission electron
microscopy (see Panel 1−1, p. 13). To avoid damage, it is
important that only a few electrons pass through each part of the
specimen, sensitive detectors are therefore deployed to capture
every electron that passes through the specimen. Much EM
specimen preparation and data collection is now fully automated
and many thousands of micrographs are typically captured, each
of which will contain hundreds or thousands of individual
molecules all arranged in random orientations within the ice.
Algorithms then sort
the particles into sets
that each contains
particles that are all
oriented in the same
direction. The
thousands of images
in each set are all
then superimposed
and averaged to
improve the signal to
noise ratio.
5 nm
This crisper two-dimensional
image set, which represents
different views of the particle,
are then combined and
converted via a series of
complex iterative steps into a
high resolution
three-dimensional structure.
Model of GroEL
(Courtesy of Gabriel Lander.)
CRYO-EM STRUCTURE OF
THE RIBOSOME
60S large ribosomal subunit at
0.25 nm resolution
path of a rRNA loop fitted
into the electron density map
Courtesy of Joachim Frank.
60S ribosomal subunits randomly
oriented in a thin film of ice
Mg 2+
G C
RNA bases
100 nm 5 nm 1 nm
Although by no means routine, big improvements in
image processing algorithms, modeling tools and sheer
computing power all mean that structures of
macromolecular complexes are now becoming attainable
with resolutions in the 0.2 to 0.3 nm range.
This resolving power now approaches that of x-ray
crystallography, and the two techniques thrive together, each
bootstrapping the other to obtain ever more useful and dynamic
structural information. A good example is the structure of the
ribosome shown here at a resolution of 0.25 nm.