LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE 595

(A) (B) (C) (D)

500 nm 500 nm 50 nm 10 nm

appear as multiple copies in the tomogram can be identified, and with a compu- Figure 9–48 EM tomography. The COP1
tational process called subtomogram averaging to reduce noise and gain structural coat mediates vesicle traffic within the
Golgi apparatus and retrograde traffic
information, molecular structures inside cells can now be obtained at a resolution
to the endoplasmic reticulum (ER) (see
of better than 2 nm (Figure 9–48). Electron microscopy now provides a robust Figures 13–4 and 13–5). EM tomography
bridge between the scale of the single molecule and that of its cellular environment. has helped visualize the details of COP1
coats in situ on buds and vesicles in rapidly
MBoC7 n9.131/9.48
Cryo-electron Microscopy Can Determine Molecular Structures frozen Chlamydomonas cells. (A) One slice
through a three-dimensional tomogram
at Atomic Resolution of a complete Golgi apparatus. (The
tomogram can be seen in Movie 9.2.)
As we saw earlier (p. 567), noise is important in light microscopy at low light lev- (B) Using the information from several
els, but it is a particularly severe problem for electron microscopy of unstained such tomograms, a portion of the Golgi
macromolecules. A protein molecule can tolerate a dose of only a few hundreds of is shown here, color coded to show ER
electrons per square nanometer without damage, and this dose is orders of mag- dark yellow, the cis vesicles yellow, the
four cis cisternae green, the four medial
nitude below what is needed to define an image at atomic resolution. cisternae red, the trans cisterna blue,
The solution is to obtain images of many identical molecules—perhaps hun- medial vesicles pink, trans vesicles light
dreds of thousands of images of individual particles—and combine them to blue, and the trans Golgi network purple.
produce an averaged image, revealing structural details that are hidden by the Ribosomes can also be seen as small gray
noise in the original images. This procedure is called single-particle reconstruc- blobs. (C) Individual slices through COP1
vesicles in the tomogram; the bottom one
tion (Panel 9–1). Before combining all the individual images, however, they must is partially uncoated. (D) By identifying
be aligned with each other. With the help of a computer, the digital images of ran- and averaging more than 10,000 COP1
domly distributed and unaligned molecules can be processed and combined to subunits on vesicles in the tomograms,
yield high-resolution reconstructions (see Movie 13.1). Although structures that a molecular structure was obtained by
have some intrinsic symmetry, such as dimers or helical repeats, are somewhat subtomogram averaging at a resolution of
2 nm. Structures of the various proteins in
easier to solve (Figure 9–49), this technique has also been used for huge macro- the COP1 coat have been solved, and they
molecular machines, such as ribosomes, that have no symmetry (see Panel 9–1). can be fitted neatly into the electron-density
Cryo-electron microscopy (cryoEM) depends crucially on very rapidly freez- envelope of the EM structure. A surface
ing the aqueous specimen to form vitreous ice, which does not allow ice crystals view of a triad of COP1 subunits on the
surface of a vesicle is shown here together
to form and therefore does not damage the specimen. A very thin (about 100 nm)
with the molecular structures (in color) of
film of an aqueous suspension of purified macromolecular complex is prepared the individual components that have been
on a microscope grid and is then rapidly frozen by being plunged into a coolant. fitted into the EM structure. (Adapted from
A special sample holder keeps this hydrated specimen at –160°C in the vacuum Y.S. Bykov et al., eLife 6:e32493, 2017,
of the microscope, where it can be viewed directly without fixation, staining, or doi 10.7554/eLife.32493.)
drying. Unlike negative staining, in which what we see is the envelope of stain
exclusion around the particle, cryoEM produces an image from the macromolec-
ular structure itself. The specialized transmission electron microscopes required
operate with much higher electron accelerating voltages than that of a routine
TEM and typically run at 300,000 V. However, as very low electron doses are
used to obtain cryoEM images, the intrinsic contrast in the images produced is
very low, and to extract the maximum amount of structural information, special
image-processing techniques must be used. Huge advances in direct electron
detectors and faster, more efficient image-processing techniques that involve
image alignment routines, motion correction, and contrast transfer function cor-
rections mean that the structures of molecules as small as 100 kilodaltons can
now be solved. The smaller the molecule, the noisier the image, and the main

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 596 Chapter 9: Visualizing Cells and Their Molecules

(A) (B) (C)

50 nm 10 nm 2 nm

Figure 9–49 CryoEM structure of microtubules. This cryoEM reconstruction of the structure of
a microtubule was helped by the intrinsic symmetry of the microtubule itself (see Figure 16–37).
This detailed model of the whole microtubule has allowed an examination of the way in which
the protofilaments interact and the way in which the whole lattice and associated proteins
are assembled. (A) CryoEM image of two intact microtubules embedded in vitreous ice. (B) A
reconstruction of the surface lattice of a single microtubule at a resolution of 0.35 nm (3.5 Å).
(C) The detailed electron-density map of the tubulin dimer extracted from the structure of the intact
microtubule. α-Tubulin is darker green, and β-tubulin is lighter green. (From E. Nogales, Mol. Biol.
MBoC7 n9.135/9.49
Cell 27:3202–3204, 2016, doi 10.1091/mbc.E16-06-0372. With permission from Elsevier.)

advantages of the method are best seen with large and sometimes flexible mac-
romolecular complexes such as viruses, ribosomes, and large integral membrane
proteins that are hard to crystallize (Figure 9–50).
A remarkable resolution of 0.12 nm (1.2 Å) has been achieved in a particularly
stable protein by cryoEM, enough to see clearly the detailed atomic structure and
to rival x-ray crystallography in resolution (Figure 9–51). Electron microscopy,
however, also has some very clear additional advantages over x-ray crystallography
(A)
(discussed in Chapter 8) as a method for macromolecular structure determination.
First, it does not require crystalline specimens. Second, it can deal with extremely receptor-binding domains
large complexes—structures that may be too large or too variable to crystallize sat-
isfactorily; for example, membrane proteins. Third, it allows the rapid analysis of
different conformations of protein machines; for example, the different states of the
F1 ATPase proton pump shown in Figure 14–31. Fourth, the glycosylation patterns
and mobile loops on the surface of proteins, which are often impossible to see in
x-ray structures, are more readily resolved in cryoEM structures. And fifth, only a
minute amount of sample is required compared with that needed to make crystals.
The analysis of large and complex macromolecular structures is helped con-
siderably if the atomic structure of one or more of the subunits is known, for

Figure 9–50 The spike protein on the SARS-CoV-2 virus. The SARS-CoV-2 virus was responsible
for the COVID-19 pandemic. Protruding from the viral membrane are many trimeric spike proteins
that mediate binding of the virus to a receptor on cells in our respiratory tract and its subsequent glycan
entry into the cell. The trimeric spike protein is a target both of our immune system and of vaccine side chains
developers. The closed conformation of the trimeric spike protein shown here, both from the top
(A) and from the side (B), was obtained from rapidly frozen intact virus particles. Spike proteins
were identified by computer from multiple tilted images of the viruses and subtomogram averaging
applied to them. The final electron-density map was determined to a resolution of 0.35 nm, good
enough for the molecular model (shown here) to be accurately fitted within its envelope, although
viral
the details of the membrane-spanning portion of the trimeric spike protein are not revealed. The membrane 2 nm
proteins are heavily N-glycosylated, and these surface glycans are shown in green, while the three
spike proteins are shown in dark green, light blue, and light brown. (PDB code: 6ZWV.) (B)

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 LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE 597

0.2 nm
(C) tyrosine arginine histidine

(A) (B)
50 nm 5 nm

Figure 9–51 Atomic resolution by

example from x-ray crystallography (Figure 9–52). Molecular models can then be cryoEM. Apoferritin is a cytosolic protein,
mathematically “fitted” or docked into the envelope of the structure determined present in almost all living organisms, that
reversibly stores iron in a nontoxic form. It
at lower resolution using the electron microscope. X-ray and cryoEM approaches is a large (474 kilodaltons) and particularly
often combine profitably together to determine molecular structures. stable molecule. Its hollow globular cage
has 24 symmetrical subunits, which means
that a structure can be determined with
Light Microscopy and Electron Microscopy
MBoC7 n9.133/9.51 Are Mutually Beneficial
relatively few particles. (A) Cryo-electron
The interior of the cell is a confusing place, with molecules crowded together in micrograph of cage-like apoferritin particles.
(B) By use of every possible new technical
the cytosol and intricate and complex membrane-bounded compartments. To advance in single-particle reconstruction,
discover which molecules are located exactly where and in which tiny vesicles the complete cryoEM structure shown here
or subcompartments of the cell is not straightforward, even with the genetically is at the remarkable resolution of 0.12 nm
encoded labels that can target almost any protein. We have seen that superreso- (1.2 Å). (C) When the known amino acid
lution light microscopy can be used to very accurately locate specific molecules sequence is modeled into the electron-
density map, clear electron densities can be
within a cell. A major disadvantage, however, of all fluorescence imaging tech- seen associated with hydrogen atoms in the
niques is that it is only the tagged molecules that are imaged—their cellular three amino acid side chains. The molecular
context remains invisible. When fluorescence imaging is combined, however, model is fitted into the final electron-density
with looking at the same specimen in the electron microscope, this correlative envelope that is shown as a gray cage.
light microscopy and electron microscopy technique, or CLEM, can allow specific (A, from T. Nakane et al., Nature 587:152–
156, 2020, doi 10.1038/s41586-020-2829-0;
target molecules to be examined in their full cellular context. Although this can B, EMD-11668; C, adapted from K.M. Yip
be achieved using fixed and sectioned material, most such approaches now use et al., Nature 587:157–161, 2020. With
rapidly frozen material to co-localize target molecules both in the light and in the permission from Nature.)

EZH2 nucleosome
Figure 9–52 PRC2, a large
histone core macromolecular machine. Polycomb
repressive complex 2 (PRC2) is a
large protein complex involved in
establishing heterochromatin and the
epigenetic regulation of gene expression
(see Figure 4–40). PRC2 interacts with a
nucleosome through the binding of the
nucleosomal DNA by one of its subunits,
EZH2, which also engages the extended
tail of histone H3 to direct its lysine 27
(K27) to the active site for methylation. The
density map of PRC2 and two essential
cofactors bound to a single nucleosome
DNA
was produced by single-particle cryo-
electron microscopy reconstruction at a
resolution of 0.35 nm. The long arm of
H3 histone tail histone H3 is shown in more detail with
the protein backbone modeled into the
density map. (Courtesy of Vignesh Kasinath
PRC2 and Eva Nogales and based on EMDB-
21707. From V. Kasinath et al., Science
371:eabc3393, 2021. With permission
lysine 27 from AAAS.)

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