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Principles & Protocols
Structural biology using Transmission Electron Microscopy (TEM)
Principles
Background of structural biology
Uniqueness of electron microscopy
Principles of TEM
Cryo-EM
Protocols
Protocols
Principles
Background of structural biology
The objective of structural biology is to understand how
a macromolecule, a complex of macromolecules, or a cellular sub-section
functions through elucidation of its three-dimensional structure. Ideally, one
attempts to obtain structural information about the specimen of interest
in its native state or under conditions that allow the adoption of
physiological state(s). The level of detail (optical resolution) of the structural
information determines what can be learned about the system under study. Optical
resolution is defined as the distance at which two features can be resolved
into two distinct entities. Take for example the cell membrane, in which the
phospholipid headgroup regions in the two leaflets of the bilayer are separated
by ~5 nm (50 Å). Depending on the resolution of the image of this cell membrane, the two
leaflets would either appear as one line (e.g. resolution of 150 Å), or as two
distinct lines (e.g. resolution of 20 Å).
Thus, in the resolution range of 50-150 Å structural information is useful in
localizing different macromolecules relative to each other within a sub-section
of the cell or within a large complex. Information in the resolution range of
20-50 Å can be used to determine the spatial relationships between macromolecular
domains. Resolutions of 5-20 Å can aid in determining the location and
inter-connectivity of secondary structure elements, such as protein or RNA helices,
within macromolecules. And finally, structural information in the resolution range
of 1-5 Å can provide information on the relative locations of residues/atoms
within a macromolecule.
If structural information spanning a resolution range of 1-150 Å can be obtained
from a macromolecular complex under physiological or near-physiological conditions,
an accurate three-dimensional model can be reconstructed. Obtaining an accurate
three-dimensional model is
the ultimate goal of structural biology, since such a model provides valuable
insights into the chemistry, molecular biology, and sub-cellular context
of the macromolecule of interest.
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Uniqueness of electron microscopy
X-ray crystallography and NMR are powerful tools for structure determination
in the resolution range of 1-5 Å of macromolecules that can crystallize, and
of small complexes(<50 kDa?), respectively. Electron microscopy, however, is
uniquely positioned in that structural information spanning resolutions from the
sub-atomic to the organismal level can be obtained through the application of the
appropriate technique(s). The unique position of electron microscopy among other
structural biology methods is outlined in the following table:
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TEM |
X-ray crystallography |
NMR |
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resolution
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2-150 Å
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1-5 Å
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1-5 Å
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sample size
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molecular to sub-cellular
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molecular to macromolecular complexes
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<50 kDa?
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structure determination
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direct visualization produces magnified image. Image contains phase and
amplitude information
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diffraction pattern yields amplitude information, phases are estimated.
Calculation of structural model from amplitude and estimated phases
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Measurement of distance constraints between nuclei. Calculation of
structural model using constraints
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study conditions
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native-like environment: in vitreous ice solution or crystalline membrane
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crystalline, often in non-physiological buffers with non-physiological
additives
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high concentrations in solution
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sample conformation
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native-like
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may be distorted due to crystal packing and/or non-physiological buffer
conditions
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native-like
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sample requisites
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depending on technique: purified in solution (native buffer),
2D crystallization, isolated sub-cellular region (whole or sectioned)
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3D crystallization often by use of non-physiological buffers or additives
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purified, isotope-labeled in solution
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Principles of TEM
The structural resolution obtainable with an imaging technique
is in the range of the wavelength of the electromagnetic or
matter waves used for the imaging. This is why the resolution
between two points using a light microscope is only within a few
hundred nanometers. The de Broglie wavelength of electrons
accelerated through a voltage of 100kV is ~0.04 Å.
However, not
even atomic resolution (<2 Å) has been achieved for biological
molecules. There are three reasons for this:
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(1) Radiation damage. The interaction
of electrons with organic matter causes damage, due to the
breaking of chemical bonds and the generation of free radicals,
which in turn cause further damage. Much of this damage is a
result of the heat generated upon the interaction of electrons
with the biological specimen and can be reduced by almost an
order of magnitude if the sample is cooled from room temperature
to liquid nitrogen temperatures. Coating the specimen with
heavy metals
(see staining)
can also protect from radiation damage.
Finally, simply reducing the dose of the electrons to ~10
electrons per Å2 (with some advocating 1 electron Å-2)
significantly reduces damage to the specimen.
(2) Low signal-to-noise ratio. Since the
elements comprising biological molecules have low atomic
weights, light and electrons interact weakly with these
molecules, resulting in a low signal-to-noise ratio. In all
imaging techniques this problem is circumvented by averaging the
signal from many identical molecules. In a regular,
predictable arrangement of molecules, such as in a crystal, this
averaging is straightforward, since all molecules are in the
same orientation. In single-particle cryo-EM every particle in
the frozen-hydrated sample is potentially in a different
orientation. Averaging in this case necessitates first the
determination of the relative orientation of each particle.
(3) Lens aberration. Aberrations in the lenses within the
electron microscope restrict the useful aperture to a range
corresponding to 1 Å resolution at best.
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Cryo-EM
When a specimen in a near-physiological environment is flash-
frozen to liquid nitrogen temperatures (150º K), the water in
the specimen turns into vitreous ice, which has properties
similar to those of liquid water, due to the rapid decrease in
temperature (for more details see
plunge freezing).
This technique for preparation of frozen-hydrated
specimens used in conjunction with
visualization by electron microscopy is known as the technique of cryo-
electron microscopy (cryo-EM). Previous to the invention
of the cryo-fixation technique, samples for visualization by EM
were prepared using negative-staining, a technique in which the
specimen is stained with a solution of heavy metal salts and
then air-dried, such that the outline of the specimen, coated
with metal, presents a high contrast to the surrounding. The
disadvantages of negative-staining are numerous: the specimen is
not in its native environment; no interior density variations
of the molecule are visualized, since the metal coats mostly the
outside; and the specimen may be distorted. In a frozen-hydrated
specimen, the specimen remains immersed in water, i.e. in
vitreous ice, internal variations in density are visible, and
there is minimal distortion of specimen shape or structure.
The use of flash-freezing the specimen and cryogenic
temperatures, in conjunction with the concept of averaging many
images of the specimen, form the basis of high-resolution
biological electron microscopy.
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Protocols
Protocols
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