Ray Crystallography Provides Structural Information

A great deal of information about the structure of cells, parts of cells, and even molecules has been obtained by light microscopy. The microscope uses a lens to focus radiation to form an image after it has passed through a specimen. However, a practical limitation of light microscopy is the limit of resolution. Radiation of a given wavelength cannot resolve structural features less than about 1/2 its wavelength. Since the shortest wavelength of visible light is around 400 nm, even the very best light microscopes have a theoretical limit of resolution of no less than 200 nm.

Because of the much shorter wavelength (0.004 nm) of the electron at the voltages normally used in the electron

Acidic

Basic

Acidic

Basic

Crystallography Myoglobin

FIGURE 23-5

Two-dimensional gel electrophoresis of 35S-methionine labeled total cell proteins from murine thymocytes. These proteins were first subjected to isoelectric focusing (direction of migration indicated by red arrow) and then the focused proteins were separated by SDS-PAGE (direction of migration indicated by blue arrow). The gel was exposed to x-ray film to detect the labeled proteins. [Courtesy of B. A. Osborne.]

FIGURE 23-5

Two-dimensional gel electrophoresis of 35S-methionine labeled total cell proteins from murine thymocytes. These proteins were first subjected to isoelectric focusing (direction of migration indicated by red arrow) and then the focused proteins were separated by SDS-PAGE (direction of migration indicated by blue arrow). The gel was exposed to x-ray film to detect the labeled proteins. [Courtesy of B. A. Osborne.]

microscope, the theoretical limit of resolution of the electron microscope is about 0.002 nm. If it were possible to build an instrument that could actually approach this limit, the electron microscope could readily be used to determine the detailed atomic arrangement of biological molecules, since the constituent atoms are separated by distances of 0.1 nm to 0.2 nm. In practice, aberrations inherent in the operation of the magnetic lenses that are used to image the electron beam limit the resolution to about 0.1 nm (1Â). This practical limit can be reached in the examination of certain specimens, particularly metals. Other considerations, however, such as specimen preparation and contrast, limit the resolution for biological materials to about 2 nm (20 Â). To determine the arrangement of a molecule's atoms, then, we must turn to x-rays, a form of electromagnetic radiation that is readily generated in wavelengths on the order of size of interatomic distances. Even though there are no microscopes with lenses that can focus x-rays into images, x-ray crystallography can reveal molecular structure at an extraordinary level of detail.

X-ray crystallography is based on the analysis of the diffraction pattern produced by the scattering of an x-ray beam as it passes though a crystal. The degree to which a particular atom scatters x-rays depends upon its size. Atoms such as carbon, oxygen, or nitrogen, scatter x-rays more than do hydrogen atoms, and larger atoms, such as iron, iodide, or mercury give intense scattering. X-rays are a form of electromagnetic waves;

as the scattered waves overlap, they alternately interfere with and reinforce each other. An appropriately placed detector records a pattern of spots (the diffraction pattern) whose distribution and intensities are determined by the structure of the diffracting crystal. This relationship between crystal structure and diffraction pattern is the basis of x-ray crystallographic analysis. Here is an overview of the procedures used:

OBTAIN CRYSTALS OF THE PROTEIN OF INTEREST. To those who have not experienced the frustrations of crystallizing proteins, this may seem a trivial and incidental step of an otherwise highly sophisticated process. It is not. There is great variation from protein to protein in the conditions required to produce crystals that are of a size and geometrical formation appropriate for x-ray diffraction analysis. For example, myoglobin formed crystals over the course of several days at pH 7 in a 3 M solution of ammonium sulfate, but 1.5 M ammonium sulfate at pH 4 worked well for a human IgG1. There is no set formula that can be applied, and those who are consistently successful are persistent, determined, and, like great chefs, have a knack for making just the right "sauce."

SELECTION AND MOUNTING. Crystal specimens must be at least 0.1 mm in the smallest dimension and rarely exceed a few millimeters in any dimension. Once chosen, a crystal is harvested into a capillary tube along with the solution from which the crystal was grown (the "mother liquor"). This keeps the crystal from drying and maintains its solvent content, an important consideration for maintaining the internal order of the specimen. The capillary is then mounted in the diffraction apparatus.

GENERATING AND RECORDING A DIFFRACTION PATTERN.

The precisely positioned crystal is then irradiated with x-rays of a known wavelength produced by accelerating electrons against the copper target of an x-ray tube. When the x-ray beam strikes the crystal, some of it goes straight through and some is scattered; sensitive detectors record the position and intensity of the scattered beam as a pattern of spots (Figure 23-6a,b).

INTERPRETING THE DIFFRACTION PATTERN. The core of diffraction analysis is the mathematical deduction of the detailed structure that would produce the diffraction pattern observed. One must calculate to what extent the waves scattered by each atom have combined to reinforce or cancel each other to produce the net intensity observed for each spot in the array. A difficulty arises in the interpretation of complex diffraction patterns because the waves differ with respect to phase, the timing of the period between maxima and minima. Since the pattern observed is the net result of the interaction of many waves, information about phase is critical to calculating the distribution of electron densities that is responsible. The solution of this "phase problem" looms as a major obstacle to the derivation of a high-resolution structure of any complex molecule.

The problem is solved by derivatizing the protein—modifying it by adding heavy atoms, such as mercury, and then obtaining crystals that have the same geometry as (are iso-

X-ray source

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X-ray beam

Diffracted beams

Crystallography Hyaluronic Acid

FIGURE 23-6

X-ray crystallography. (a) Schematic diagram of an x-ray crystallographic experiment in which an x-ray beam bombards the crystal and diffracted rays are detected. (b) Section of x-ray diffraction pattern of a crystal of murine IgG2a. (c) Section from the electron-density map of murine IgG2a. [Part (a) from L. Stryer, 1995, Biochemistry, 4th ed.; parts (b) and (c) courtesy of A. McPherson.]

FIGURE 23-6

X-ray crystallography. (a) Schematic diagram of an x-ray crystallographic experiment in which an x-ray beam bombards the crystal and diffracted rays are detected. (b) Section of x-ray diffraction pattern of a crystal of murine IgG2a. (c) Section from the electron-density map of murine IgG2a. [Part (a) from L. Stryer, 1995, Biochemistry, 4th ed.; parts (b) and (c) courtesy of A. McPherson.]

morphous with) those of the underivatized protein. The diffraction pattern of the isomorphous crystal is obtained and compared with that of the native protein. Usually, armed with a knowledge of the diffraction patterns of two or more isomorphous heavy-atom derivatives, the phases for the native protein can be calculated by reference to the characteristic diffraction patterns generated by heavy-atom landmarks. The phases established, it is possible to move on to a calculation of the distribution of electron density. This is accomplished by Fourier synthesis, a mathematical treatment particularly suited to the analysis of periodic phenomena such as those involving waves. In this case, it is used to compute the distribution of electron density along the x, y, and z axes within a unit cell of the crystal. The deduced electron density can then be visualized on a computer (Figure 23-6c).

DERIVATION OF THE STRUCTURE. The resolution of a model depends upon a number of factors. First of all, the ultimate resolution possible is set by the quality of the crystal and the internal order of the crystal. Even the highest-quality crystals have a degree of internal disorder that establishes a limit of resolution of about 2 A. Second, a factor of paramount importance is the number of intensities fed into the Fourier synthesis. A relatively small number of spots may produce a low-resolution (6 A) image that traces the course of the polypeptide chain but provides little additional structural information. On the other hand, the processing of data provided by tens of thousands of spots allows the tracing of very detailed electron-density maps. Provided one knows the amino-acid sequence of the protein, such maps can guide the construction of high-resolution, three-dimensional models. Amino-acid sequence data is necessary because it can be difficult, and in some cases impossible, to unambiguously distinguish among some amino-acid side chains on even the most detailed electron-density maps.

Since 1960, when the first detailed structures of proteins were deduced, the structures of many thousands of proteins have been solved. These range from small and (relatively) simple proteins such as lysozyme, consisting of a single polypep-tide chain, to poliovirus, an 8,500,000 dalton, stunningly complex nucleoprotein made up of RNA encased by multiple copies of four different polypeptide subunits. Of particular importance to immunologists are the large number of immu-nologically relevant molecules for which detailed crystal structures are now available. These include many immunoglob-ulins, most of the major and minor proteins involved in the MHC and T-cell-receptor complexes, and many other important immunological macromolecules, with new structures and structural variants appearing every month.

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Responses

  • john
    Why is the theoretical limit of resolution for an electron microscope 0.002nm equation?
    8 years ago
  • Paulina Thompson
    Why electron microscopes has limit of resolution of 0.002 nm?
    8 years ago
  • tuija pohjonen
    Can xray crystallography determine total protein structure?
    8 years ago
  • barbara
    What information is obtained from xray crystallography?
    8 years ago

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