December 19, 2012 | 4
We’re having this month’s Scicurious Guest Writer a little early, to make sure he gets some exposure and to avoid the holiday rush! Please welcome Satchal Erramilli!!
In the summer of 1912, a young man and his father worked feverishly to interpret the results of a German physicist. The physicist, future Nobel Laureate Max von Laue, had recently observed the behavior of X-rays when exposed on a crystal, and was struggling to describe the interference of X-ray waves that resulted. Though the solution eluded him, the discovery, which soon would give birth to one of the most important techniques in science, came at an exciting time. The 19th and early 20th century marked an unusually active and competitive era in science; prior to this, it was believed that much of scientific theory had been conclusively worked out, particularly in the physical sciences. However, in the early 1900s, a number of brilliant researchers made startling breakthroughs that showed we were just scratching the surface of our scientific knowledge. It was in this vigorous environment that the young man, William Lawrence Bragg, then a graduate student on his summer break, and his father, William Henry Bragg, a mathematician and physicist, scrambled to understand a series of observations made by von Laue.
Upon his return to graduate school at Cambridge, where he was studying mathematics, the younger Bragg had made his breakthrough, and his doctoral adviser, the Nobel Laureate J.J. Thomson, presented his results on 11 November 1912 to the Cambridge Philosophical Society. Bragg had deduced what von Laue had struggled with: how individual spots on an X-ray diffraction pattern related to the atomic structure of the crystal that scattered them. Bragg’s formulation, now known as Bragg’s Law, successfully identified these positions. This meant, in essence, that, by crystallizing molecules and exposing the crystals to X-rays, the structures of individual molecules could be determined. The result was a seminal moment in the history of science, the birth of what remains today the most accurate technique to determine molecular structures: X-ray Crystallography. For his part in discovering X-ray diffraction from crystals, von Laue won the Nobel Prize in Physics in 1914, while both W.H. and W.L Bragg won the Nobel Prize in Physics in 1915 for the formulation. Since then hundreds of thousands of molecular structures have been determined via X-ray crystallography, with important consequences for physics, chemistry, and biology. The centenary gives us an opportunity to look back on not just Bragg but on the many great researchers who followed.
First off, what is crystallography, and how do X-rays come into play? After William Roentgen’s discovery of X-rays in the late 19th century, many scientists worked to better understand the behavior of X-rays. Among them was von Laue, who observed the diffraction of X-rays by salt crystals and diamonds. If you’ve never observed diffraction, it is the phenomenon of scattering radiation by a repeated array. You can try this at home with a basic laser pointer and a razor blade; pointing this towards a blank wall will produce a pattern that arises from the diffraction of visible light by the edge of the blade. Then, align a second blade with the edge of the first such that the edges form a narrow slit, and shine the laser through it. The two edges form a grating that results in the diffraction of visible light. The key is in the slit; as long as it is of similar magnitude to the wavelength of radiation, it will successfully diffract the light.
In a crystal, the bonds between atoms serve as tiny gratings. What von Laue observed was that X-rays, with wavelengths similar to the size of molecular bonds, could be scattered by atoms, and the resulting pattern reflected the arrangement of atoms in the crystal. How does that pattern arise? In one respect, diffraction can be thought of as the scattering of X-rays, which consist of photons, by atoms in a molecule, much like the deflection of balls during a game of pool. However, X-rays, like all types of radiation, behave as both particles and waves. In this context, diffraction can be considered the bending of radiation around matter. If you’ve ever observed seawater as it flows into a harbor, you will see the waves diffract, or spread out, as they pass through the narrow opening of the harbor.
Additionally, the diffracted X-rays, because of the three-dimensional arrangement of atoms in the crystal, will travel different distances on their way to the X-ray detector, which records the pattern. A property of all waves is interference: waves can add constructively (increase in intensity) or destructively (decrease in intensity). Based on the position of atoms within the crystal, the X-rays will interfere with each other to varying degrees by the time they arrive at the detector, producing a pattern with spots of varying intensity. Bragg formulated a way to deduce the position of atoms within a crystal, giving birth to the idea that molecular structures can be determined from crystals.
But why is a crystal required? Why couldn’t a single molecule diffract the X-rays? A crystal, essentially a repeating unit of a particular molecule, significantly amplifies the signal of the diffraction pattern. Based on the limits of current X-ray technology, a single molecule is insufficient to produce a robust enough signal from its diffraction pattern.
X-ray crystallography has greatly improved our knowledge of physics, chemistry, and organic chemistry, but its impact on biology has been enormous. At its most fundamental level, biology is the interaction of many tiny molecular players, invisible to even the most robust light microscopes. For example, proteins are the workhorses of the cell, tiny machines that carry out nearly every significant biological process imaginable. X-ray crystallography provides the means to determine what these “invisible” molecules look like and to help answer some fundamental questions about their roles in cells, and how they carry these roles out.
By the late 1940s, the now Nobel Prize-winning W. L. Bragg had become well established at Cavendish Laboratories in Cambridge and, after studying salt crystals and diamonds, became interested in biological macromolecules. In the early 1950s, one of his former students established his own lab at Cavendish and would soon change the face of molecular biology research forever. The aforementioned student, Max Perutz, had worked out how to produce protein crystals for use in X-ray diffraction studies. Upon completing his PhD, Perutz remained at Cambridge, where he and his students worked out how to determine the more complicated protein structures using crystals. Many inorganic and small organic compounds contain tens or hundreds of atoms; proteins can consist of tens or hundreds of thousands of atoms. As a result, data from protein crystals require special processing to determine structures. Working out this method allowed Perutz and colleagues to determine the first protein structure, of hemoglobin, in 1959. Perutz shared the Nobel Prize in Chemistry in 1962 for his efforts.
By the mid-1960s, Michael Rossmann and David Blow, both contributors to Perutz’s Nobel Prize winning work, were pioneering the modern version of crystallography, developing computer programs that allowed automated processing of protein crystallographic data. This greatly sped up the determination of protein structures, and combined with technical advances in X-ray and computational sciences, has made the process near routine. It took Perutz and colleagues more than six years to determine the structure of hemoglobin after publishing their method for data processing. By the mid-1970s, about a dozen protein structures had been determined. Nowadays, several thousands of protein structures are determined each year, and nearly seventy thousand have been deposited in the Protein Data Bank.
Protein structure determination is only part of X-ray crystallography’s impact on biology. Though we now take for granted the role of DNA in the transmission of genetic information, how this information could be stored in molecular form was largely unknown until the mid-20th century. That DNA was the means of storing genetic information only began to gain widespread acceptance in the 1940s. This part of the story takes us back to Cavendish Laboratories (home to Bragg and Perutz), where Francis Crick and James Watson worked as researchers. Though neither worked on the structure of DNA initially, they were both obsessed about it, spending long hours debating how it might look. Meanwhile, another researcher at Cambridge, Rosalind Franklin, collected X-ray diffraction data of DNA fibers, using a technical offshoot of crystallography (while DNA fibers are not crystals, they form a regular repeating array of nucleic acids, and so diffract X-rays). With permission from W.L. Bragg and Max Perutz, but not from Franklin, Francis and Crick studied her diffraction data, and correctly solved the structure before she could do so. In 1953, they published the structure of the DNA double helix in a seminal Nature paper, and won the Nobel Prize in 1962. Though Franklin collected the data that Watson and Crick used for their result, she never received co-authorship on their subsequent publication, and after her passing due to ovarian cancer in 1958, could not be part of the Nobel award. This controversy marred what was otherwise a monumental result in the history of science, one that essentially gave birth to the field of molecular biology.
The contributions of crystallography to biology were not limited to DNA and proteins. In the 1960s and ‘70s, Hugh Huxley, also at Cambridge, used X-ray diffraction to understand the basis of muscle contraction. Muscles are essentially filaments of the protein myosin and actin (also known as thick and thin filaments, respectively), bundled into repeating arrays (thus enabling diffraction) wherein myosin fibers can form thousands of cross-bridges with actin to help shorten fibers, promoting muscle fiber contraction.
X-ray diffraction studies allowed for the observation of structural changes in an isolated muscle fiber during contraction, providing tremendous insight into this process. Beginning in the late ‘70s, and continuing through this decade, much work was done on the ribosome – the cellular powerhouse responsible for taking the message from a cell’s genetic material and interpreting it to produce proteins. It took many long years to crystallize and solve the structure of the ribosome, but the results provided insight into how the ribosome functions, as well as into how antibiotics interact with it (because bacterial and eukaryotic ribosomes differ, it is possible to target them specifically using bactericides, such as erythromycin and streptomycin). The successful completion of this work led to the award of the 2009 Nobel Prize in Chemistry to three of the signature researchers in this field: Tom Steitz, Venki Ramakrishnan, and Ada Younath.
In fact, over two-dozen awards have been given to works associated with crystallography since von Laue’s discovery in the early 20th century. In the 2000s alone, six of the 24 awards in either Physics or Chemistry have gone to crystallographers, including the most recent, given to Robert Lefkowitz and Brian Kobilka for their work on the structures of G-protein Coupled Receptors (GPCRs). GPCRs are among the most important molecules in the cell; residing at the interface of the cell and its external environment, GPCRs are involved in the perception of nearly every relevant stimuli in humans, including light, touch, and taste. The work by Lefkowitz, Kobilka, and many others has provided critical insight into how GPCRs function, and much of that insight can be traced back to crystallographic results.
Sadly, the next great milestone in X-ray crystallography’s history may end up its obituary. Work is being carried out the world over to produce powerful enough X-rays to successfully diffract from individual molecules and get a robust enough signal to determine structures. For instance, pioneering work has recently begun at Stanford’s X-ray Free Electron Laser, a 2-mile long source that produces such powerful beams it outstrips its predecessors several fold. On 29th November, a long-awaited paper was published in Science, where researchers described the first novel structure determined using this X-ray source, utilizing crystals that were several times smaller than those normally required for diffraction studies. With improving technology, this could set the stage for single-molecule diffraction. However things may evolve, let us not forget Bragg’s discovery, and the innumerable contributions in the one hundred years thereafter, that enabled us to finally “see” molecules and forever changed how we think about life.
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Woolfson, Michael (1997), An Introduction to X-Ray Crystallography (Cambridge)
Ladd, M., Palmer, A. (2003), Structure Determination by X-Ray Crystallography (Springer)
Drenth, J. (1999), Principles of Protein X-ray Crystallography (Springer)
Thomas, J.M., Centenary: The birth of X-ray crystallography, Nature 491: 186-87 (2012)
Baker, M., Structural biology: the gatekeepers revealed, Nature 465: 823-826 (2010)
Weber, A., Franzini-Armstrong, C., Hugh E. Huxley: birth of the sliding filament model of muscle contraction, Trends in Cell Biology 12: 243-245 (2002)
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As far as bio: Satchal Erramilli is a doctoral student in the lab of Cynthia Stauffacher at Purdue University, where he studies the biophysics of membrane proteins, mainly because it sounds cool. He is definitely gruntled. You can follow him @erudeite or read more of his work at satchal.blogspot.com.
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