Principles & 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. back to top

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 organism 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:

TEM X-ray crystallography NMR

resolution 2-150 A 0.8-5 A atomic

sample size molecular to sub-cellular molecular to macromolecular complexes <50 kDa

structure determination Direct visualization produces magnified image. Image contains phase and amplitude information. Measurement of distance constraints between nuclei. Calculation of structural model using constraints. Diffraction pattern yields amplitude information, phases are estimated. Calculation of structural model from amplitude and estimated phases.

study conditions Native-like environment: in vitreous ice solution or crystalline membrane. Crystalline, often in non-physiological buffers with non-physiological additives. High concentrations in solution.

sample conformation native-like May be distorted due to crystal packing and/or non-physiological buffer conditions. native-like

sample requisites depending on technique: purified in solution (native buffer), 2D crystallization, isolated sub-cellular region (whole or sectioned) 3D crystallization often by use of non-physiological buffers or additives. Purified, isotope-labeled in solution.

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:

(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 [[CemProtocStaining][staining]) can also protect from radiation damage. Finally, simply reducing the dose of the electrons to ~10 electrons per A2 (with some advocating 1 electron A-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 1A resolution at best. back to top

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.

-- KakoliMitra - 11 Feb 2009