The Structures of Life
Chapter 2: X-ray Crystallography: Art Marries Science
How would you examine the shape of something too small to see in even the most powerful microscope? Scientists trying to visualize the complex arrangement of atoms within molecules have exactly that problem, so they solve it indirectly. By using a large collection of identical molecules—often proteins—along with specialized equipment and computer modeling techniques, scientists are able to calculate what an isolated molecule would look like.
The two most common methods used to investigate molecular structures are X-ray crystallography (also called X-ray diffraction) and nuclear magnetic resonance (NMR) spectroscopy. Researchers using X-ray crystallography grow solid crystals of the molecules they study. Those using NMR study molecules in solution. Each technique has advantages and disadvantages. Together, they provide researchers with a precious glimpse into the structures of life.
More than 85 percent of the protein structures that are known have been determined using X-ray crystallography. In essence, crystallographers aim high-powered X-rays at a tiny crystal containing trillions of identical molecules. The crystal scatters the X-rays onto an electronic detector like a disco ball spraying light across a dance floor. The electronic detector is the same type used to capture images in a digital camera.
After each blast of X-rays, lasting from a few seconds to several hours, the researchers precisely rotate the crystal by entering its desired orientation into the computer that controls the X-ray apparatus. This enables the scientists to capture in three dimensions how the crystal scatters, or diffracts, X-rays.
The intensity of each diffracted ray is fed into a computer, which uses a mathematical equation called a Fourier transform to calculate the position of every atom in the crystallized molecule.
The result—the researchers' masterpiece—is a three-dimensional digital image of the molecule. This image represents the physical and chemical properties of the substance and can be studied in intimate, atom-by-atom detail using sophisticated computer graphics software.
An essential step in X-ray crystallography is growing high-quality crystals. The best crystals are pure, perfectly symmetrical, three-dimensional repeating arrays of precisely packed molecules. They can be different shapes, from perfect cubes to long needles. Most crystals used for these studies are barely visible (less than 1 millimeter on a side). But the larger the crystal, the more accurate the data and the more easily scientists can solve the structure.
Using X-ray crystallography, scientists can study enormous viruses that contain several hundred proteins. Mavis Agbandje-McKenna uses the technique to investigate how viruses infect cells. Read about her unusual scientific and personal journey from a rural village in Nigeria to the University of Florida in Gainesville, at https://publications.nigms.nih.gov/findings/
Crystallographers grow their tiny crystals in plastic dishes. They usually start with a highly concentrated solution containing the molecule. They then mix this solution with a variety of specially prepared liquids to form tiny droplets (1-10 microliters). Each droplet is kept in a separate plastic dish or well. As the liquid evaporates, the molecules in the solution become progressively more concentrated. During this process, the molecules arrange into a precise, three-dimensional pattern and eventually into a crystal—if the researcher is lucky.
Sometimes, crystals require months or even years to grow. The conditions—temperature, pH (acidity or alkalinity), and concentration—must be perfect. And each type of molecule is different, requiring scientists to tease out new crystallization conditions for every new sample.
Even then, some molecules just won't cooperate. They may have floppy sections that wriggle around too much to be arranged neatly into a crystal. Or, particularly in the case of proteins that are normally embedded in oily cell membranes, the molecule may fail to completely dissolve in the solution.
Some crystallographers keep their growing crystals in air-locked chambers, to prevent any misdirected breath from disrupting the tiny crystals. Others insist on an environment free of vibrations—in at least one case, from rock-and-roll music. Still others joke about the phases of the moon and supernatural phenomena. As the jesting suggests, growing crystals remains one of the most difficult and least predictable parts of X-ray crystallography. It's what blends art with the science.
Although the crystals used in X-ray crystallography are barely visible to the naked eye, they contain a vast number of precisely ordered, identical molecules. A crystal that is 0.5 millimeters on each side contains around 1,000,000,000,000,000 (or 1015) medium-sized protein molecules.
When the crystals are fully formed, they are placed in a tiny glass tube or scooped up with a loop made of nylon, glass fiber, or other material depending on the preference of the researcher. The tube or loop is then mounted in the X-ray apparatus, directly in the path of the X-ray beam. The searing force of powerful X-ray beams can burn holes through a crystal left too long in their path. To minimize radiation damage, researchers flash-freeze their crystals in liquid nitrogen.
Crystal photos courtesy of Alex McPherson, University of California, Irvine
In order to measure something accurately, you need the appropriate ruler. To measure the distance between cities, you would use miles or kilometers. To measure the length of your hand, you would use inches or centimeters.
Crystallographers measure the distances between atoms in angstroms. One angstrom equals one ten-billionth of a meter, or 10-10m. That's more than 10 million times smaller than the diameter of the period at the end of this sentence.
The perfect "rulers" to measure angstrom distances are X-rays. The X-rays used by crystallographers are approximately 0.5 to 1.5 angstroms long-just the right size to measure the distance between atoms in a molecule. There is no better place to generate such X-rays than in a synchrotron.
Ribosomes make the stuff of life. They are the protein factories in every living creature, and they churn out all proteins ranging from bacterial toxins to human digestive enzymes.
To most people, ribosomes are extremely small—tens of thousands of ribosomes would fit on the sharpened tip of a pencil. But to a structural biologist, ribosomes are huge. They contain three or four strands of RNA and more than 50 small proteins. These many components work together like moving parts in a complex machine—a machine so large that it has been impossible to study in structural detail until recently.
In 1999, researchers determined the crystal structure of a complete ribosome for the first time. The work was a technical triumph for crystallography. Even today, the ribosome remains the largest complex structure obtained by crystallography. (Some larger virus structures have been determined, but the symmetry of these structures greatly simplified the process.)
This initial snapshot was like a rough sketch that showed how various parts of the ribosome fit together and where within a ribosome new proteins are made. Today, researchers have extremely detailed images of ribosomes in which they can pinpoint and study every atom.
In addition to providing valuable insights into a critical cellular component and process, structural studies of ribosomes may lead to clinical applications. Many of today's antibiotics work by interfering with the function of ribosomes in harmful bacteria while leaving human ribosomes alone. A more detailed knowledge of the structural differences between bacterial and human ribosomes may help scientists develop new antibiotic drugs or improve existing ones.
Imagine a beam of light 30 times more powerful than the Sun, focused on a spot smaller than the head of a pin. It carries the blasting power of a meteor plunging through the atmosphere. And it is the single most powerful tool available to X-ray crystallographers.
This light, one of the brightest lights on earth, is not visible to our eyes. It is made of X-ray beams generated in large machines called synchrotrons. These machines accelerate electrically charged particles, often electrons, to nearly the speed of light, then whip them around a huge, hollow metal ring.
Synchrotrons were originally designed for use by high-energy physicists studying subatomic particles and cosmic phenomena. Other scientists soon clustered at the facilities to snatch what the physicists considered an undesirable byproduct—brilliant bursts of X-rays.
The largest component of each synchrotron is its electron storage ring. This ring is actually not a perfect circle, but a many-sided polygon. At each corner of the polygon, precisely aligned magnets bend the electron stream, forcing it to stay in the ring (on their own, the particles would travel straight ahead and smash into the ring's wall). Each time the electrons' path is bent, they emit bursts of energy in the form of electromagnetic radiation.
This phenomenon is not unique to electrons or to synchrotrons. Whenever any charged particle changes speed or direction, it emits energy. The type of energy, or radiation, that particles emit depends on the speed the particles are going and how sharply they are bent. Because particles in a synchrotron are hurtling at nearly the speed of light, they emit intense radiation, including lots of high-energy X-rays.
Synchrotrons are prized not only for their ability to generate brilliant X-rays, but also for the "tunability" of these rays. Scientists can actually select from these rays just the right wavelength for their experiments.
In order to determine the structure of a molecule, crystallographers usually have to compare several versions of a crystal—one pure crystal and several others in which the crystallized molecule is soaked in, or "doped" with, a different heavy metal, like mercury, platinum, or uranium.
Because these heavy metal atoms contain many electrons, they scatter X-rays more than do the smaller, lighter atoms found in biological molecules. By comparing the X-ray scatter patterns of a pure crystal with those of various metal-containing crystals, the researchers can determine the location of the metals in the crystal. These metal atoms serve as landmarks that enable researchers to calculate the position of every other atom in the molecule.
But when using X-ray radiation from the synchrotron, researchers do not have to grow multiple versions of every crystallized molecule—a huge savings in time and money. Instead, they grow only one type of crystal that contains the chemical element selenium instead of sulfur in every methionine amino acid. They then "tune" the wavelength of the synchrotron beam to match certain properties of selenium. That way, a single crystal serves the purpose of several different metal-containing crystals. This technique is called MAD, for Multi-wavelength Anomalous Diffraction.
Using MAD, the researchers bombard the selenium-containing crystals three or four different times, each time with X-ray beams of a different wavelength—including one blast with X-rays of the exact wavelength absorbed by the selenium atoms. A comparison of the resulting diffraction patterns enables researchers to locate the selenium atoms, which again serve as markers, or reference points, around which the rest of the structure is calculated.
The brilliant X-rays from synchrotrons allow researchers to collect their raw data much more quickly than when they use traditional X-ray sources, which are small enough to fit on a long laboratory table and produce much weaker X-rays than do synchrotrons.What used to take weeks or months in the laboratory can be done in minutes at a synchrotron. But then the data still must be analyzed, refined, and corrected before the protein can be visualized in its three-dimensional structural splendor.
The number and quality of molecular structures determined by X-ray diffraction has risen sharply in recent years, as has the percentage of these structures obtained using synchrotrons. This trend promises to continue, due in large part to new techniques like MAD and to the matchless power of synchrotron radiation.
In addition to their role in revealing molecular structures, synchrotrons are used for a variety of applications, including to design computer chips, to test medicines in living cells, to make plastics, to analyze the composition of geological materials, and to study medical imaging and radiation therapy techniques.
“Science is like a roller coaster. You start out very excited about what you're doing. But if your experiments don't go well for a while, you get discouraged. Then, out of nowhere, comes this great data and you are up and at it again.”
That's how Juan Chang describes the nature of science. He majored in biochemistry and computer science at the University of Texas at Austin. He also worked in the UT-Austin laboratory of X-ray crystallographer Jon Robertus.
Chang studied a protein that prevents cells from committing suicide. As a sculptor chips and shaves off pieces of marble, the body uses cellular suicide, also called "apoptosis," during normal development to shape features like fingers and toes. To protect healthy cells, the body also triggers apoptosis to kill cells that are genetically damaged or infected by viruses.
By understanding proteins involved in causing or preventing apoptosis, scientists hope to control the process in special situations-to help treat tumors and viral infections by promoting the death of damaged cells, and to treat degenerative nerve diseases by preventing apoptosis in nerve cells. A better understanding of apoptosis may even allow researchers to more easily grow tissues for organ transplants.
Baylor College of Medicine
Chang was part of this process by helping to determine the X-ray crystal structure of a protein that scientists refer to as ch-IAP1. He used biochemical techniques to obtain larger quantities of this purified protein. The next step will be to crystallize the protein, then to use X-ray diffraction to obtain its detailed, three-dimensional structure.
Chang came to Texas from a lakeside town on the northwest tip of Venezuela. He first became interested in biological science in high school. His class took a field trip to an island off the Venezuelan coast to observe the intricate ecological balance of the beach and coral reef. He was impressed at how the plants and animals—crabs, insects, birds, rodents, and seaweed—each adapted to the oceanside wind, waves, and salt.
About the same time, his school held a fund drive to help victims of Huntington's disease, an incurable genetic disease that slowly robs people of their ability to move and think properly. The town in which Chang grew up, Maracaibo, is home to the largest known family with Huntington's disease. Through the fund drive, Chang became interested in the genetic basis of inherited diseases.
His advice for anyone considering a career in science is to "get your hands into it" and to experiment with work in different fields. He was initially interested in genetics, did biochemistry research, and is now in a graduate program at Baylor College of Medicine. The program combines structural and computational biology with molecular biophysics. He anticipates that after earning a Ph.D., he will become a professor at a university.
What is meant by the detailed, three-dimensional structure of proteins?
What is X-ray crystallography?
Give two reasons why synchrotrons are so valuable to X-ray crystallographers.
What is a ribosome and why is it important to study?