What is X-ray Crystallography?

X-ray crystallography is a complex and dynamic field that has been associated with several of science’s major breakthroughs in the second half of the 20^th century. Using X-ray crystal data, Drs. James Watson and Francis Crick were able to determine the helical structure of DNA in 1953. More recently, in 1998, Dr. Peter Kim, was able to determine the structure of GP120, a key protein responsible for the HIV infection process. This structure has provided scientists the means for producing new drugs, preventing the deadly HIV infection.

X-ray crystallography is a scientific technique that is very similar to medical X-rays. Instead of using an X-ray machine and peering into a person, X-ray crystallography uses X-ray radiation to determine the shape of a molecule that has been crystallized into a solid. All solids have a subatomic lattice structure that gives the molecule its solid properties. A well-known crystalline solid that has a simple lattice structure is table salt, NaCl. Understanding how a molecule packs into a lattice structure and how X-ray radiation “reflects” inside that lattice, allows scientists to determine the structure of a chemical or biological molecule.

Figure 1: In X-ray crystallography, an X-ray beam is diffracted by a crystal. The diffraction pattern can be recorded as spots where the diffracted X-rays strike a photographic plate.
Historical Background of X-ray Crystallography.

The English physicist Sir William Henry Bragg (picture shown below) pioneered the determination of crystal structure by X-ray diffraction methods, for which he and his son William Lawrence Bragg received the 1915 Nobel Prize for physics. After Wilhelm Roentgen discovered X rays in 1895, Bragg began a lifelong investigation of the nature of radiation, principally X rays but also alpha and beta particles and gamma rays. After the discovery of the diffraction of X rays by crystals in 1912, Bragg and his son, William L., derived Bragg's law, which relates the wavelength of X rays to the glancing angle of reflection. In 1913 the elder Bragg built the first X-ray spectrometer, which he initially used to study X-ray spectral distributions. Within several years they were able to use this instrument and Bragg's law to derive the structure of crystals and show the exact positions of atoms. Subsequently, they demonstrated that the properties and behavior of a large variety of substances can be related to the position of their constituent atoms.

William Lawrence Bragg went on to become director of the Cavendish Laboratory in Cambridge England. It was at this lab, while he as director, in the early 1950's that J.D. Watson and F.H.C. Crick, using the X-ray diffraction techniques that Bragg pioneered, deduced the double helical structure of Deoxyribonucleic acid (DNA).

http://ep.llnl.gov/bep/science/12/tXray.html

Clinton J. Davisson (picture shown below) was an American experimental physicist and Bloomington High School graduate who shared the Nobel Prize for Physics in 1937 with George P. Thomson of England for discovering that electrons can be diffracted like light waves, thus verifying the thesis of Louis de Broglie that electrons behave both as waves and as particles.

Davisson received his doctorate from Princeton University and spent most of his career at the Bell Telephone Laboratories. He began his research there on the emissions of electrons from a metal in the presence of heat and later helped develop the electron microscope.

Then, in 1927, Davisson and Lester H. Germer found that a beam of electrons, when reflected from a metallic crystal, shows diffraction patterns similar to those of X-rays and other electromagnetic waves. This discovery verified quantum mechanics' understanding of the dual nature of subatomic particles and proved to be useful in the study of nuclear, atomic, and molecular structure, including X-ray crystallography.

http://search.eb.com/nobel/micro/161_29.html

What can X-ray Crystallography tell us?

X-ray crystal data can give a variety of information. Based on X-ray data the order in which the atoms are attached can be determined, including bond angles and bond lengths. From bond lengths, atomic radii and periodic trends can be inferred. Additionally, the arrangement of the atoms and their orientation in 3D space can sometimes give a researcher an idea of the chemical mechanism for forming that molecule.

X-ray Crystallography and DNA.

In 1953, two young scientists, James Watson and Francis Crick, proposed the double helical structure of DNA. These conclusions were based on X-ray crystallographic results provided by Rosalind Franklin and base composition studies from Erwin Chargaff. Earlier attempts to solve the structure of DNA using X-ray crystallography were conducted by William Astbury in 1938, but to limited success. The DNA samples were found to be not pure enough. However, Astbury was able to predict that the bases were stacked like a roll of pennies, separated by a distance of 3.4Å (3.4 x 10^-10 m).

By 1953 Rosalind Franklin, working at the Cavendish Laboratory in Cambridge, England, had obtained improved X-ray crystal results. Based on purified samples of DNA, Franklin was able to confirm Astbury’s 3.4Å results, as well as, DNA’s helical nature (shown at the right). Unfortunately, she never provided a definitive structure for DNA. Her analysis paved the way for two interpretations of the raw X-ray crystallographic data, Linus Pauling’s triple helix and Watson and Crick’s double helical model. In the end, Watson and Crick’s double helical model proved to be correct and they were awarded the Nobel Prize in Medicine in 1962.

Klug, W.S., Cummings, M.R. Concepts of Genetics, Prentice Hall 2000.

X-ray Crystallography and Protein Structures.

Human Immunodeficiency Virus (HIV)

In 1998, researchers at the Dana-Farber Cancer Institute and Columbia University made a major breakthrough in Human Immunodeficiency Virus (HIV) research. Researchers were able to isolate, identify and crystallize an important protein involved in the viral invasion process. Through their research, this protein, GP120, demonstrates a two lock mechanism. This discovery proved invaluable by showing researchers that GP120 has a region found in all HIV variants that was always the same. Further, this indicated the cavity in all HIV variants were potentially susceptible to highly specific drug regimes.

Figure 2: Topographical representations of CD4 and GP120 respectively. The GP120 protein has been rotated to show the point of interaction.

Figure 3: Docking of the CD4 (left) and GP120 (right).

As Figure 2 and 3 illustrates, a phenylalanine amino acid residue sticks out from the end of the CD4. This phenylalanine residue inserts itself into a small cavity within the GP120 binding site, which is visible in Figure 2. Notice how the colors line up, blue with blue and red with red, especially around the immediate vicinity of the cavity. The phenylalanine acts as a key, while the cavity on GP120 is acting like a lock.

Ebola

The Ebola virus (shown at the right) has struck fear throughout the world and rightfully so. This virus has graced the pages of magazines and newspapers for several decades and has even been the basis of a Hollywood blockbuster. Ebola has become synonymous with the word outbreak, but how much do scientists really know. Within the past decade, major breakthroughs in Ebola research have come as a result of advances in Human Immunodeficiency Virus (HIV). It has recently been shown that both Ebola and HIV are evolutionarily related.

Ebola is a virus that causes hemorrhagic fever, made evident through profuse bleeding from every orifice of the patient. Ebola’s natural host remains to be identified, however recent evidence indicates bats may serve as that host.

X-ray crystallographic analysis indicates Ebo-74, a protein found on the outer membrane of the Ebola virus, is similar to other structures found in HIV, SIV (HIV for monkeys) and Influenza (Figure 4).

Figure 4: Figure 4 illustrates the similarity of Ebola’s Ebo-74 protein to like proteins found in HIV and influenza.

Ebo-74 looks like three hairpins next to one another. Figure 5b shows a pictorial representation of Ebo-74, while 5a illustrates only one of the three hairpin structures. This hairpin structure is similarly found in the previously mentioned viruses and reinforces previous claims of an evolutionary relationship.

Figure 5: Figure 5a (left) shows one of the three hairpin proteins associated with Ebo-74 complex while Figure 5b (right) illustrates the trimer-of-hairpins complex that makes up the Ebo-74 protein.

It has been speculated that the trimer-of-hairpins acts as a spring loaded protein. Through structural changes, this spring-loaded protein “spikes” itself into the target cell’s outer membrane. This action brings the two membranes, the viral membrane and the target membrane, close enough for their membranes to fuse, becoming one. It is at this point infection is eminent. However, the exact mechanism is unknown for this “harpoon” theory.

With the similarity of Ebola firmly established to many other viruses, conclusions can be formulated by making comparisons to viruses that have been better studied. Although the information is merely speculation at this time, the “harpoon” theory remains a viable mechanistic approach to the membrane fusion process. This mechanism has proven to have drug intervention possibilities in other viral systems and may be the source of future drug research for effective Ebola treatment.

Interesting Applications of X-ray Crystallography at ISU.

X-ray crystallography is used extensively in the ISU Chemistry Dept. Not only does X-ray crystallography serve as a teaching aid and course for upper level chemistry courses, but it also provides an invaluable resource to many of the faculty’s personal research. Dr. Shawn Hitchcock studies pseudoephedrine derived ring systems (left) as potential drug systems. Pseudoephedrine is the active ingredient found in over-the-counter antihistamines such as Sudafed and Nyquil. The X-ray structures of this system provide detailed information regarding the bond distance and angles associated with the individual atoms and the two carbon/oxygen double bond units. Through the introduction of a variety of different chemical groups (R), the angle of the two carbon/oxygen double bond units (carbonyls) can be studied. The larger the chemical group, the more distorted the angle of the carbonyls becomes (Figure 7). Knowing such structural details helps to predict the properties, including potential drug characteristics, and the mechanism for synthesizing other similar molecules.

Figure 6: Pseudoephedrine derived ring system where R = -CH₃.

Figure 7: Side view of four pseudoephedrine derivatives where R = -CH₃, -CH₂CH₃,

-C₆H₅ and -C(CH₃)₃