X-RAY CRYSTALLOGRAPHY BASIC INFORMATION AND TUTORIALS
What is X-Ray Crystallography?
X-ray crystallography is the most important method for determining protein structure. X-ray crystallography is a technique by which an x-ray beam is scattered upon contact with a crystallized protein. The pattern of scattering can be used to determine the relative position of the atoms in the crystal.
The quality of an x-ray crystal structure is described in terms of its resolution. Resolution is listed in units of angstroms (Å). A high resolution for an x-ray structure might be 0.5–2.0 Å with a lower number indicating better resolution.
Once the data has been processed, the positions of nonhydrogen atoms appear as spheres in three-dimensional space in an image called an electron density map. The electron density map shows atoms as spheres.
Figure 9.1 shows the impact of resolution on the readability of an electron density map. Image (a) has a resolution of 0.62 Å. Atoms are nearly distinct and separate from other atoms. Image (b) has a resolution of 1.2 Å. Atoms are still clear but somewhat larger.
As the resolution drops to 2.0 Å (c), the spheres of the individual nuclei become unclear. The feature that now dominates the electron density map is the peptide backbone. Once the resolution drops to 3.0 Å (d) and 4.0 Å (e), single atom branches off the backbone are nearly imperceptible. At 5.0 Å (f), folds of the backbone can mistakenly appear to merge together.
Solving an x-ray structure requires correctly assigning atoms in the protein to match the electron density map. Before solving a structure, an x-ray crystallographer already has the sequence of the target protein from the molecular biology team.
The x-ray crystallographer begins to solve the structure by aligning the most obvious residues, such as the aromatic rings, into the electron density map. Less obvious residues then fall into place as possibilities become more limited. If an electron density map has a low resolution (6–8 Å), then only general features such as a-helices or b-sheets may be clear. The confident placement of individual residues is not possible.
A protein will generally have one or more random coil regions in its overall structure. Random coils, as the name suggests, do not have a regular, folded structure and undergo rapid, continuous conformational changes. These regions appear indistinct even in an otherwise high resolution x-ray structure. Fortunately, binding and active sites generally occupy ordered protein regions that are amenable to characterization by x-ray crystallography.
Knowing the structure and shape of the binding site of a target allows the discovery team to make informed decisions about molecules that will bind to the target. Is the binding site deep or shallow? Are the amino acid residues in the binding site polar, nonpolar, or charged?
The properties of the binding site determine the optimal, complementary properties needed in a drug that will interact with the binding site. Often molecules can be cocrystallized with an enzyme target. The x-ray structure of the complex shows both how a compound fills the binding pocket and precisely where a compound may be modified to better fill the active site.
TOP TEN SERENDIPITOUS DISCOVERIES IN CHEMISTRY
1. Archimedes: Streaking Around
Archimedes was a Greek mathematician who lived in the third century BCE. Hero, the king of Syracuse, gave Archimedes the task of determining whether Hero’s new gold crown was composed of pure gold, which it was supposed to be, or whether the jeweler had substituted an alloy and pocketed the extra gold.
Archimedes figured that if he could measure the density of the crown and compare it to that of pure gold, he’d know whether the jeweler had been dishonest. But although he knew how to measure the weight of the crown, he couldn’t figure out how to measure its volume in order to get the density.
Needing some relaxation, he decided to bathe at the public baths. As he stepped into the full tub and saw the water overflow, he realized that the volume of his body that was submerged was equal to the volume of water that overflowed.
He had his answer for measuring the volume of the crown. Legend has it that he got so excited that he ran home naked through the streets, yelling, “Eureka, eureka!” (I’ve found it!).
2. Vulcanization of Rubber
Rubber, in the form of latex, was discovered in the early 16th century in South America, but it gained little acceptance because it became sticky and lost its shape in the heat. Charles Goodyear was trying to find a way to make the rubber stable when he accidentally spilled a batch of rubber mixed with sulfur on a hot stove.
He noticed that the resulting compound didn’t lose its shape in the heat. Goodyear went on to patent the vulcanization process, the chemical process used to treat crude or synthetic rubber or plastics to give them useful properties such as elasticity, strength, and stability.
3. Molecular Geometry
In 1884, the French wine industry hired Louis Pasteur to study a compound left on wine casks during fermentation — racemic acid. Pasteur knew that racemic acid was identical to tartaric acid, which was known to be optically active — that is, it rotated polarized light in one direction or another. When
Pasteur examined the salt of racemic acid under a microscope, he noticed that two types of crystals were present and that they were mirror images of each other. Using a pair of tweezers, Pasteur laboriously separated the two types of crystals and determined that they were both optically active, rotating polarized light the same amount but in different directions.
This discovery opened up a new area of chemistry and showed how important molecular geometry is to the properties of molecules.
4. Mauve Dye
In 1856, William Perkin, a student at The Royal College of Chemistry in London, decided to stay home during the Easter break and work in his lab on the synthesis of quinine. (I guarantee you that working in the lab isn’t what my students do during their Easter break!)
During the course of his experiments, Perkin created some black gunk. As he was cleaning the reaction flask with alcohol, he noticed that the gunk dissolved and turned the alcohol purple — mauve, actually. This was the synthesis of the first artificial dye.
5. Kekulé: The Beautiful Dreamer
Friedrich Kekulé, a German chemist, was working on the structural formula of benzene, C6H6, in the mid-1860s. Late one night, he was sitting in his apartment in front of a fire. He began dozing off and, in the process, saw groups of atoms dancing in the flames like snakes.
Then, suddenly, one of the snakes reached around and made a circle, or a ring. This vision startled Kekulé to full consciousness, and he realized that benzene had a ring structure. Kekulé’s model for benzene paved the way for the modern study of aromatic compounds.
6. Discovering Radioactivity
In 1856, Henri Becquerel was studying the phosphorescence (glowing) of certain minerals when exposed to light. In his experiments, he’d take a mineral sample, place it on top of a heavily wrapped photographic plate, and expose it to strong sunlight.
He was preparing to conduct one of these experiments when a cloudy spell hit Paris. Becquerel put a mineral sample on top of the plate and put it in a drawer for safekeeping.
Days later, he went ahead and developed the photographic plate and, to his surprise, found the brilliant image of the crystal, even though it hadn’t been exposed to light. The mineral sample contained uranium. Becquerel had discovered radioactivity.
7. Finding Really Slick Stuff: Teflon
Roy Plunkett, a DuPont chemist, discovered Teflon in 1938. He was working on the synthesis of new refrigerants. He had a full tank of tetrafluoroethylene gas delivered to his lab, but when he opened the valve, nothing came out. He wondered what had happened, so he cut the tank open.
He found a white substance that was very slick and nonreactive. The gas had polymerized into the substance now called Teflon. It was used during World War II to make gaskets and valves for the atomic bomb processing plant. After the war, Teflon finally made its way into the kitchen as a nonstick coating for frying pans.
8. Stick ’Em Up! Sticky Notes
In the mid-1970s, a chemist by the name of Art Frey was working for 3M in its adhesives division. Frey, who sang in a choir, used little scraps of paper to keep his place in his choir book, but they kept falling out.
At one point, he remembered an adhesive that had been developed but rejected a couple years earlier because it didn’t hold things together well. The next Monday, he smeared some of this “lousy” adhesive on a piece of paper and found that it worked very well as a bookmark — and it peeled right off without leaving a residue. Thus was born those little yellow sticky notes you now find posted everywhere.
9. Growing Hair
In the late 1970s, minoxidil, patented by Upjohn, was used to control high blood pressure. In 1980, Dr. Anthony Zappacosta mentioned in a letter published in The New England Journal of Medicine that one of his patients using minoxidil for high blood pressure was starting to grow hair on his nearly bald head.
Dermatologists took note, and one — Dr. Virginia Fiedler-Weiss — crushed up some of the tablets and made a solution that some of her patients applied topically. It worked in enough cases that you now see Minoxidil as an over-the counter hair-growth medicine.
10. Sweeter Than Sugar
In 1879, a chemist by the name of Fahlberg was working on a synthesis problem in the lab. He accidentally spilled on his hand one of the new compounds he’d made, and he noticed that it tasted sweet. He called this new substance saccharin.
James Schlatter discovered the sweetness of aspartame while working on a compound used in ulcer research. He accidentally got a bit of one of the esters he’d made on his fingers. He noticed its sweetness when he licked his fingers while picking up a piece of paper.
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