100 Years of X-ray Crystallography

"Our ignorance of solids is very nearly complete," lamented physical chemist H. C. Jones in his 1913 book A New Era in Chemistry. "We do not know what is the formula of solid sodium chloride or rock salt; or of solid water or ice; and we have no reliable means at present of finding out these simplest matters."

Credit: Wikimedia Commons

William Henry Bragg

Credit: SPL/Science Source

William Lawrence Bragg

Credit: Wikimedia Commons

Max von Laue

Jones didn't realize how quickly this was about to change.

In fact, father and son team William Henry Bragg and William Lawrence Bragg had already begun chipping away at this ignorance, by using X-ray crystallography to reveal atomic structure.

The younger Bragg had been captivated by a result reported by German physicist Max von Laue. Pass an X-ray beam through the atomic lattice of a crystal and the X-rays should diffract, von Laue had surmised, yielding a characteristic interference pattern of bright spots on a photographic plate. His collaborators collected the first such diffraction pattern from a copper sulfate crystal in April 1912. The result seemed to settle what had been a source of hot debate: X-rays behave like waves.

But explaining the origin of the pattern of spots was another matter entirely. Enter the younger Bragg. "I can remember the exact spot on the Backs [the riverside in Cambridge, England] where the idea suddenly leapt into my mind that Laue's spots were due to the reflection of X-ray pulses by sheets of atoms in the crystal," Lawrence later wrote. X-rays, he surmised, could therefore be used to reveal molecules' atomic structure.

The idea, encapsulated in what we now call Bragg's law, proved transformative. Inspired by his son's theory, Henry built the first X-ray spectrometer, which the pair quickly put to use to record the reflections of X-rays from a series of crystals.

Their structure of sodium chloride and those of related alkaloid salts revealed that crystals could be made up of repeating atomic lattices rather than molecular ones. Their structure of diamond provided long-sought experimental support for the theory that carbon is tetrahedral.

"It was a wonderful time," Lawrence later recalled. "Like discovering a new goldfield where nuggets could be picked up on the ground, with thrilling new results every week."

That is, until World War I stopped their work together. Lawrence headed off to war while Henry turned his talents to submarine detection. But the discipline the Braggs founded, X-ray crystallography, has continued to thrill during the past century, giving scientists a window on the atomic world. In honor of 2014's International Year of Crystallography, we've highlighted a few of our favorite X-ray crystal structures—ones that answered pressing chemical questions of their day.

Credit: Wikimedia Commons

Interference pattern observed by von Laue and collaborators using a photographic plate. The central spot is from the unscattered X-ray beam. The dark spots result when X-rays scattering from different layers of a ZnS crystal interfere constructively.

Credit: Science Museum, London, Wellcome Images

The Braggs' spectrometer.

According to chemistry lore, the idea for the ring-based structure of benzene came to German chemist Friedrich August Kekulé in a daydream, in which he envisioned a snake eating its own tail. Although Michael Faraday first isolated benzene in 1825, chemists had not nailed down its structure.

Kekulé's dream led him to propose that the structure contained a six-membered ring of carbon atoms with alternating single and double bonds—a theory that he published in 1865.

But questions still remained. In what configuration was this ring? Was it puckered or bowed or flat? Did the molecule have three distinct double bonds?

Most chemists subscribed to the theory that benzene was flat, but it wasn't until crystallographer Kathleen Lonsdale entered the picture in 1929 that the mystery was finally solved.

Credit: Hulton-Deutsch Collection/Corbis

Lonsdale in her lab in 1948.

Lonsdale obtained her bachelor's degree in physics at age 19 in 1922. She was the first woman tenured professor at University College London and the first woman president of the British Association for the Advancement of Science. In 1956, she was named Dame of the British Empire.

In an essay about her life, Lonsdale wrote, "It had been my intention to give up scientific research work and settle down to become a good wife and mother, but my husband would have none of it."

In the 1920s, she set up a crystallography lab in the physics department at Leeds University, where her husband was also a scientist.

Lonsdale's historic relationship with hexamethylbenzene began when she was given some crystals of the compound. Unlike benzene itself, hexamethylbenzene has just one molecule per unit cell, making it easier to distinguish the orientation of the molecule's central benzene ring. Lonsdale's X-ray crystallography experiments showed without doubt that the benzene ring was not only flat, but also had an evenly distributed cloud of electrons, sharing the three double bonds (Nature 1928, DOI: 10.1038/122810c0).

Because the benzene ring is the foundation of aromatic compounds, the discovery made possible innumerable inroads into their chemistry.

Lonsdale's discovery was also notable in that chemists were just starting to make progress in using X-ray crystallography to determine the structures of organic molecules. Organic molecules, with their relative lack of symmetry, were much harder to study than symmetric inorganic lattices. The first X-ray structure of an organic molecule, hexamethylenetetramine, had been reported a mere 5 years earlier, in 1923.

In the late 1920s, the structure of water in its crystalline form—ice—was an area of much debate. "Data on the subject in the literature are very conflicting," wrote William H. Barnes at the time, noting that textbooks and other resources featured varying structures.

A Canadian scientist who used a fellowship to visit London's Royal Institution to work with crystallography pioneer William H. Bragg, Barnes wound up publishing the definitive structure of ice formed under natural environmental conditions. He showed conclusively that the oxygens are arranged in a planar hexagon, with the hydrogens located between the oxygens (Proc. R. Soc. Lond. A 1929, DOI: 10.1098/rspa.1929.0195). The hexagons assemble into parallel sheets, giving ice a structure somewhat akin to graphite.

Credit: Proc. R. Soc. Lond. A

Barnes's drawing of the structure of ice from his 1929 paper.

Barnes's "technique and skill were just amazing," says Bruce M. Foxman, a chemistry professor at Brandeis University. "It's just really some of the most beautiful experimental work."

Barnes didn't work at just one temperature—he characterized ice from 0 to –183 °C, using a liquid-air cryostat. Setting up the experiment would have been challenging, Foxman says, let alone analyzing the diffraction pattern. "Let's not forget that there weren't any calculators or computers; this was all looking at intensities and matching strong for strong, weak for weak," Foxman emphasizes. The work also predated understanding of hydrogen bonding.

We now know that the structure determined by Barnes drives many of the properties of frozen water, from snowflakes to sea ice.

Credit: Proc. R. Soc. Lond. A

A photograph of an ice sample studied by Barnes.

To form a snowflake, you start with a "little round ball of ice," says Kenneth G. Libbrecht, a professor of physics at California Institute of Technology. Water molecules add preferentially to the edges of ice's hexagonal sheets. As the sheets get larger, the corners stick out further, so they encounter more water molecules and grow ever faster. "Pretty soon you have a hexagon with branches and side branches," and the stunning beauty of a snowflake, Libbrecht says.

Sea ice grows similarly, with the planes poking down into the water, says Pat Langhorne, a professor of physics at New Zealand's University of Otago. The structure of ice also explains its unique lower density as a solid than a liquid. Ice's ability to float on liquid water in turn gives it a key role in moderating Earth's climate by reflecting sunlight.

"This material is not ordinary in any way," Langhorne says. "The properties that it has are critical to life."

By today's standards, penicillin is a pretty small molecule. But when Dorothy Crowfoot Hodgkin determined the antibiotic's structure in 1945, it was the largest molecule yet to have its structure solved by X-ray crystallography.

That X-ray structure helped settle the debate over the structure of penicillin. After scientists figured out in 1943 that penicillin contains a sulfur atom, they had two main hypotheses of its structure. One hypothesis, advocated primarily by British organic chemist and future Nobelist, Robert Robinson, thought the structure consisted of two five-membered rings connected by a single bond, the thiazolidine-oxazolone structure. The other side, led by Edward P. Abraham and Ernst B. Chain at the University of Oxford and Robert Burns Woodward at Harvard, was convinced that the structure consisted of a four-membered β-lactam ring fused to a five-membered ring.

The chemical evidence, much of it based on degradation, thermochemical, and spectroscopic studies, supported the β-lactam structure, but Robinson's prominence and persuasiveness kept the controversy going. The crystal structure drove the point home. In May 1945, Hodgkin's X-ray diffraction data showed that the β-lactam model was right. Even with the X-ray data, however, Robinson remained unconvinced.

Credit: Science Museum, London

The X-ray crystal structure of penicillin definitively showed that the β-lactam model, as shown in this model Hodgkin built, was correct.

Hodgkin and her colleagues grew crystals of sodium, potassium, and rubidium benzylpenicillin, the form then known as penicillin G in the U.S. and penicillin II in the U.K. The electron density maps didn't form an isomorphous series, which made solving the structure more difficult. Hodgkin needed to draw projections of the different structures to the same scale and then rotate them until many of the peaks coincided.

Because the wartime work on penicillin was considered secret, the structure was not published until December 1945 and even then in tentative language. The unambiguous structure declaration was finally published four years later in The Chemistry of Penicillin, a behemoth 1,000-plus-page monograph featuring sometimes contradictory chapters written by many of the people involved in the wartime penicillin efforts of the U.S. and the U.K.

The X-ray structure of penicillin was "a considerable achievement," Chain said in his Nobel Lecture in 1946.

"For the first time the structure of a whole molecule has been calculated from X-ray data, and it is the more remarkable that this should have been possible in the case of a substance having the complexity of the penicillin molecule," he said. Hodgkin's Nobel, honoring her structural work on not just penicillin but also the even more structurally complex vitamin B-12, came years later, in 1964.

In 1951, Johannes M. Bijvoet, a crystallographer at Utrecht University in the Netherlands, reported an X-ray crystal structure that changed the course of organic chemistry. Bijvoet's absolute structure of sodium rubidium (+)-tartrate tetrahydrate marked the first time stereochemistry had been determined by experiment (Nature 1951, DOI: 10.1038/168271a0). Organic chemists could finally associate the molecular properties of an optically active compound with its absolute configuration.

The work revealed that German chemist Emil Fischer had guessed correctly when arbitrarily defining glucose's D configuration as the (+)-isomer. This meant that the configurations of the hundreds of organic compounds that had been determined based on Fischer's arbitrary assignment were also correct. Organic compounds no longer had to be tediously synthesized from a molecule of known chirality for chemists to be certain of their configuration.

Credit: IUCr

Bijvoet

Bijvoet's work also marked a key advance in X-ray crystallography. In conventional X-ray diffraction, crystallographers measure only the amplitudes of the diffraction. This information reveals interatomic distances but not phase information, which is necessary to determine absolute configuration. Bijvoet got around this problem by using a technique called anomalous X-ray scattering. The method had been used previously to determine the absolute structure of the inorganic compound zinc sulfide. Bijvoet was the first to apply it to an organic molecule.

The strategy involves using an X-ray wavelength that just excites a single heavy atom—in Bijvoet's case, rubidium—to extract phase information. Anomalous X-ray scattering is still widely used today.

Bijvoet became interested in using X-ray crystallography for structure determination of organic molecules in the early 1940s, says Martin Lutz, a structural chemist at the Bijvoet Center for Biomolecular Research at Utrecht University. At the time, Fritz Kögl, an organic chemist there, had a theory that nonnatural D-amino acids caused cancer. He needed a method to distinguish D-amino acids from L-amino acids, Lutz notes. Bijvoet recalled the structure determination of zinc sulfide by X-ray crystallography. "He was able to transfer this inorganic knowledge to the organic world," Lutz says.

A year after publishing the structure, Bijvoet moved his lab to a converted villa, making his private home part of the same building. His students called it the Crystal Palace. He lived there until he retired from Utrecht in 1962. Bijvoet passed away in 1980, at the age of 88.

Sometimes what science needs to move forward is a hearty dose of disbelief. Such was the case with ferrocene, the beloved sandwich-shaped molecule that's been credited with ushering in the modern age of organometallic chemistry.

On Dec. 15, 1951, Peter L. Pauson and Thomas J. Kealy, two chemists at Duquesne University in Pittsburgh, reported a new type of organo-iron compound (Nature, DOI: 10.1038/1681039b0). They suggested their remarkable yellow crystals consisted of two cyclopentadiene moieties, each making a single bond to a central iron atom. It was a modest report, taking up less than a page, but it certainly raised some eyebrows.

Unbeknown to Pauson and Kealy, British Oxygen Co. chemists Samuel A. Miller, John A. Tebboth, and John F. Tremaine had made the exact same compound through an entirely different synthetic route. In a paper that appeared shortly after Pauson and Kealy's, they too proposed a C₅H₅–Fe–C₅H₅ structure (J. Chem. Soc. 1952, DOI: 10.1039/jr9520000632).

Harvard University chemists Robert Burns Woodward and Geoffrey Wilkinson read Pauson and Kealy's paper and believed their proposed structure to be incorrect. In short order, they proposed an alternative structure in which the iron sat sandwiched between two parallel cyclopentadiene rings (J. Am. Chem. Soc. 1952, DOI: 10.1021/ja01128a527). Ernst Otto Fischer, of the Technische Hochschule in Munich, made a similar proposal (Z. Naturforsch. B: Chem. Sci. 1952, 7, 377).

Credit: Nature

Electron-density projections of ferrocene.

"I think it is difficult today to appreciate just how surprising, unorthodox, even revolutionary, this structure must have appeared to most chemists," writes crystallographer Jack D. Dunitz in an essay on ferrocene he wrote in the 1993 book Organic Chemistry: Its Language and Its State of the Art. Dunitz, who in 1951 was a research fellow at the University of Oxford, recalls that his own reaction was one of extreme skepticism. "I thought: What a nerve these Harvard chemists have! To publicly put forward such a structure on such scanty evidence!"

He was not alone. Marshall D. Gates, an assistant editor for the Journal of the American Chemical Society, wrote to Woodward, "We have dispatched your communication to the printers but I cannot help feeling that you have been at the hashish again."

Dunitz shared his disbelief with theoretical chemist Leslie E. Orgel, who was also at Oxford. The two decided to persuade an organic chemist they knew to prepare crystals of ferrocene, which Dunitz used to solve the structure. "Extraordinary as it seemed to me, the Harvard proposal was correct," Dunitz recalls. "There was no doubt about it."

Dunitz published the structure along with Orgel's explanation of ferrocene's stability based on molecular orbital theory (Nature 1953, DOI: 10.1038/171121a0). Independently, Pennsylvania State University researchers Philip F. Eiland and Raymond Pepinsky also used crystallography to confirm the sandwich structure (J. Am. Chem. Soc. 1952, DOI: 10.1021/ja01139a527). "That was the marvelous thing about crystal structure analysis," Dunitz remarks. "When it worked, the result had a satisfying definiteness about it."

It is one of the most famous molecular structures ever determined, the now iconic DNA double helix. Although X-ray diffraction played a role in its solution by James D. Watson and Francis H. C. Crick in 1953, it was not an X-ray crystal structure. That wouldn't come for another 20 years from the laboratory of Alexander Rich at Masschusetts Institute of Technology.

The data that inspired Watson and Crick's breakthrough wasn't from a crystal; Rosalind Franklin and her student R. G. Gosling produced diffraction patterns from fibrous DNA. Franklin, a serious crystallographer, didn't even know that Watson and Crick had access to her X-ray diffraction data on two distinct forms of DNA, dubbed A and B. The data had come to them in a somewhat roundabout way through the agency of Max F. Perutz, who directed the University of Cambridge lab in which they both worked.

The data clearly indicated to them that B-DNA was a double helix and provided key information about its dimensions. They also knew from the work of Erwin Chargaff that, regardless of the provenance of the DNA, adenine and thymine bases and guanine and cytosine bases were always present in approximately equal amounts.

Credit: A. Barrington Brown/Science Source

Watson (left) and Crick in 1953 with their model of part of a DNA molecule.

That information was enough for them to build their model, which suggested immediately how DNA encoded genetic information and how it could replicate (Nature 1973, DOI: 10.1038/171737a0). It eventually earned them the 1962 Nobel Prize in Physiology or Medicine, which they shared with Maurice Wilkins, a colleague of Franklin's. Franklin had died in 1958 at the age of 37 from ovarian cancer.

Wilkins and Franklin and Watson and Crick weren't the only scientists chasing the structure of DNA. Looming over them was the towering figure of Linus C. Pauling, who only three years earlier had used X-ray diffraction data and model building to correctly deduce the α-helix and β-sheet secondary structures of proteins.

Many people have suggested that, had Pauling had access to Franklin's X-ray data, he would have very likely deduced the correct structure of DNA. Pau­ling, however, didn't have access to Franklin's data. Because of Pauling's political activism, the U.S. State Department denied him a passport in 1952 and prevented him from making a planned trip to London.

In the 20 years that followed Watson and Crick's Nature publication, X-ray crystal structures of simple DNA structures had called into question their hypothesis. In May 1973, however, Rich and coworkers reported they had solved the X-ray crystal structure of the dinucleoside phosphate aden­osyl-3',5'-uridine phosphate, noting that it "crystallizes in the form of a right handed antiparallel double helix with Watson-Crick hydrogen bonding between uracil and adenine" (Nature, DOI: 10.1038/243150a0). Other DNA X-ray crystal structures would follow, but Rich's team had settled the question once and for all.

In the early 20th century, doctors prescribed a diet of beef liver for those suffering from pernicious anemia. But they had only a limited understanding of why the stuff cured the otherwise deadly disease. The key turned out to be vitamin B-12, which E. Lester Smith's group at Glaxo and another team at Merck isolated independently in 1948. Chemists immediately wanted to determine its chemical formula and structure.

So Smith sent crystals of the vitamin to University of Oxford chemist Dorothy Crowfoot Hodgkin, a pioneering crystallographer who had solved the structure of penicillin. She and her research team immediately began analyzing the ruby-red crystals.

With approximately 100-nonhydrogen atoms, vitamin B-12 was the most complex molecule yet tackled by crystallographers. Hodgkin was excited to find that it contained a cobalt atom. She had used the presence or substitution of heavy atoms to help define earlier structures, and she realized cobalt could play a similar role. She collaborated with Princeton University chemist John G. White and his team to solve the structure over about six years.

Credit: C&EN Archives

Hodgkin demonstrates the structure of vitamin B-12 with a model.

Jenny P. Glusker, now an emeritus professor at Fox Chase Cancer Center, was a graduate student in Hodgkin's lab at the time. She remembers collecting and estimating the intensities of the diffraction data by eye and finding the position of the cobalt atom. "Each electron density map took six weeks, day and night, with a whole roomful of IBM cards," she says. The team built a three-dimensional model with wire for bonds and wax balls for atoms—which often melted during hot summers, Glusker recalls. Kenneth N. Trueblood at UCLA helped accelerate their efforts by doing calculations with an early supercomputer.

The complete structure, published in 1956, was surprising and complex (Nature, DOI: 10.1038/178064a0). The cobalt at the center of the molecule was part of a ring structure similar to porphyrin, called the corrin ring. In 1961, Hodgkin and P. Galen Lenhert published the structure of the vitamin's coenzyme form (Nature, DOI: 10.1038/192937a0). This was the first time biochemists had seen a carbon-cobalt bond, paving the way for advances at the interface of inorganic and biological chemistry.

At the time, some scientists minimized the sophistication required to solve B-12's structure, Glusker says. "They thought a crystallographer was just a technician who pressed a button, and out came the answer."

The work led to the total synthesis of vitamin B-12 by Harvard University chemist Robert Burns Woodward, Albert Eschenmoser of ETH Zurich, and collaborators in 1972. Doctors can now give shots of the vitamin to patients with pernicious anemia. In 1964 Hodgkin won the Nobel Prize in Chemistry for determining this structure and others, including penicillin. She went on to solve the structure of insulin.

On Nov. 5, 1965, about five months after publishing the first-ever X-ray crystal structure of an enzyme in the journal Nature, David C. Phillips gave a public lecture recounting the feat.

The audience, out for some Friday night entertainment, marveled over the molecular models Phillips had brought with him to the lecture theater at London's Royal Institution. Hanging from the ceiling was a 32-foot-long string of the enzyme's 129 amino acids. And on the counter next to Phillips was the same string, only folded into a globular shape a few feet across. The folding, Phillips explained, locked the amino acids together in a way that gave the enzyme its catalytic activity.

"It must have been one of 'the' moments in time for a scientist," says the University of British Columbia's Stephen G. Withers of the historic lecture. "As a longtime enzymologist, I can only imagine what it was like for Phillips and his colleagues to see inside one of these beasts for the first time ever."

Credit: Royal Institution Archives

Phillips unveils the molecular model of lysozyme (on table in front of him) to the public during a historic lecture at the Royal Institution in 1965. The enzyme's linear sequence hangs from the ceiling.

The enzyme whose anatomy the researchers laid bare—lysozyme—was long known to kill bacteria by chewing through peptidoglycans that strengthen the microbes' outer cell walls. Alexander Fleming, who would go on to discover penicillin, had identified lysozyme in 1922, after he splashed some drops of nasal mucus onto a plate of bacteria and noticed the organisms stop growing. Lysozyme, scientists would later learn, exists inside the body as a natural defender against bacterial invaders.

But it wasn't understood how the enzyme accomplished this task until Phillips and his team determined their 2-Å-resolution X-ray structure of lysozyme from chicken egg whites (Nature 1965, DOI: 10.1038/206757a0). At the time, this version of lysozyme was easier to isolate than human lysozyme.

The 3D structure revealed lysozyme's active site, a cleft in the enzyme's globular landscape where bacterial peptidoglycans fit snuggly and had their glycosidic bonds cleaved. After Phillips's graduate student at the Royal Institution, Louise N. Johnson, succeeded in crystallizing lysozyme with some amino-sugar units of peptidoglycan chains, the team proposed a mechanism of action for the enzyme.

According to Withers, who refined that mechanism years later (Nature 2001, DOI: 10.1038/35090602), "They did a remarkably good job of fitting everything together," given that X-ray structures were all calculated by hand back then.

At the time, "a lot was known about how reactions were catalyzed in solution," Withers continues. The lysozyme structure, he adds, helped scientists understand how those same types of reactions were carried out inside enzymes.

Today, crystallographers routinely use lysozyme to calibrate X-ray instruments, and professors use it to train future generations of structural chemists and biologists.

The three-dimensional structure of transfer RNA (tRNA) was determined by Alexander Rich and coworkers at Massachusetts Institute of Technology in 1973, but it didn't come easy. "The story of how you get there, from knowing nothing to knowing everything, is a lot of hard work," says Rich, now a spry 89-year-old living in Woods Hole, Massachusetts.

tRNA mediates translation of the genetic code into the amino acid sequence of a protein. It picks up amino acids inside cells and delivers them to the protein-assembling ribosome factory, where messenger RNA gene transcripts guide their incorporation into growing protein chains.

Credit: Science

This L-shaped structure of tRNA was determined by Rich and coworkers in 1973.

"We used to think amino acids bound directly to mRNA" before getting strung into proteins, Rich says. Then Francis H. C. Crick "got the idea that maybe there's a small amino acid-bound molecule that binds to mRNA," and that turned out to be tRNA. "Then the question was, How does this molecule carry out its function?"

Robert W. Holley of Cornell University and others predicted that the sequence of tRNA could be folded into a three-leaf cloverleaf pattern. But proving it by X-ray analysis was challenging.

Indeed, it took Rich and coworkers about three years to grow suitable crystals. The key turned out to be a polyamine called spermine, which they used to stabilize the tRNA molecule's fold, making it possible to form a stable crystal lattice.

In the simple 3D diffraction pattern of tRNA that Rich's group obtained in 1971, they found evidence for the presence of a short helix consistent with Holley's cloverleaf formulation.

Credit: Science

Three-leaf prediction of how tRNA's sequence could fold.

To proceed further, Rich and coworkers needed to find an end-terminus of tRNA to properly "phase" the diffraction pattern, a process required to obtain detailed high-resolution structures. Analyzing an osmium salt of tRNA did the trick.

But solving the tRNA structure remained difficult. For example, "We had an antiquated data collection system we had developed ourselves, where you collect data point by point, one at a time," Rich says. Using those data to trace the molecule's folding pattern "was very slow and laborious."

They finally succeeded in 1973 (Science, DOI: 10.1126/science.179.4070.285).

"We found the chain had unusual folding in which the cloverleaf shape was preserved but was folded over to form an L shape," Rich says. "No one had anticipated that the molecule would be organized in this fashion."

Later that year, Aaron Klug of the University of Cambridge and coworkers reported substantially the same structure, based on a similar years-long effort.

"We in the end could explain in great detail the chemistry of the tRNA molecule and the way its folding facilitated its biological behavior," Rich says. But it took years of work to get to that point.

Ask chemists about the molecule that ushered in the new era of X-ray crystallography studies of short-lived excited-state molecules, and many would point to the nitroprusside ion.

X-ray crystallography had always been a tool for the study of molecules in stasis, their ground-state structures locked in a crystal lattice.

But as X-ray sources grew more sophisticated over the decades, the possibility of using the technique to study the structure of molecules in their excited state was not lost on chemists. They knew they would likely be able to learn immeasurable amounts about how molecules change their structure as they're about to react or even during their reaction.

The nitroprusside ion was believed to have a photoinduced excited state with a lifetime of more than two hours at low temperatures. In the early 1990s, chemistry professor Philip Coppens, at the University at Buffalo, SUNY, reasoned this state's long life would make the molecule an ideal candidate for a fledgling excited-state X-ray crystallographic study.

He succeeded, and in a 1994 paper, his team reported the structure, which was hailed as the first excited-state structure obtained by X-ray crystallography (J. Am. Chem. Soc., DOI: 10.1021/ja00091a030).

However, as Coppens tells C&EN, the story isn't so straightforward. He and his colleagues soon discovered that the photoinduced metastable state of nitroprusside was not, in fact, in an excited electronic state—it was actually the product of a photoinduced rearrangement, in which the NO group had bound differently to the transition metal.

Credit: Nancy J. Parisi/U at Buffalo, SUNY

Coppens

Nevertheless, the structure set the stage for using X-ray crystallography to examine dynamics. New synchrotron facilities, such as the Advanced Photon Source, offered bright, short X-ray pulses, which could capture the microseconds or less timescales of many excited states.

In fact, biochemists were already beginning to take advantage of synchrotron facilities to study the dynamics of "soft crystals," which are composed of biomacromolecules such as enzymes that have a lot of solvent inside. Such efforts have revealed more global dynamics, such as CO dissociation from myoglobin, but without atomic-scale resolution.

Coppens and others also began using synchrotron facilities to probe atomic-scale dynamics. Finally, in 2002, Coppens's group reported what they say was the real first X-ray crystallographic excited-state structure of a molecule: the binuclear platinum ion [Pt₂(P₂O₅H₂)₄]^4–, which has an excited state of only 50 microseconds at 17 K (Acta Crystallogr. A, DOI: 10.1107/s0108767301017986).

The Pt-Pt bond contracted by 0.28 Å when excited. A few years later, the Coppens lab discovered a much larger contraction, of 0.85 Å, in the Rh-Rh bond of the [Rh₂(1,8-diisocyano-p-menthane)₄]²⁺ ion, which had a lifetime of only 11.7 microseconds (Chem. Commun. 2004, DOI: 10.1039/b409463h).

Since then, atomic-resolution studies of excited-state crystals have proliferated. And new X-ray free-electron lasers, such as the Linac Coherent Light Source at the SLAC National Accelerator Laboratory can produce beams a billion times brighter than traditional synchrotron sources with femtosecond-timescale pulses—promising unprecedented exploration of chemical dynamics.

It's the most squirm-inducing scene in Pulp Fiction: panicked hit man John Travolta must revive mob wife Uma Thurman from a heroin overdose. So he plunges a syringe loaded with adrenaline into her heart. The film premiered in 1994. At the time, scientists lacked a precise picture of how the century-old drug worked.

The picture is clearer today, thanks to the 2011 X-ray crystal structure of adrenaline's target, the β₂ adrenergic receptor, together with the receptor's signaling partner (Nature, DOI: 10.1038/nature10361; C&EN, Aug. 1, 2011, page 9). This receptor is one of a large family of proteins called G protein-coupled receptors (GPCRs). These proteins crisscross the cell membrane seven times, transmitting messages from hormones, neurotransmitters, and even light. They are targets for as many as 50% of drugs on the market.

Credit: Nobel Foundation

For many years, researchers only had GPCR cartoons like this one to go on.

The first X-ray crystal structure of a GPCR appeared in 2000, with many more beginning in 2007. But without a G protein partner in the structure, researchers were only part of the way to seeing how signals from outside the cell get transmitted inside. The 2011 work suggests that the receptor functions by engaging the C-terminus of the G protein, tugging on it, and interfering with the region of the G protein that binds an important intracellular messenger—the nucleoside guanosine diphosphate.

Credit: Mark Lopez/Argonne National Laboratory

Kobilka

Stanford University's Brian K. Kobilka, who led the structure quest, shared the 2012 Nobel Prize in Chemistry. He says that GPCR structures in general and this structure in particular could not have been obtained without technological advances from many labs. These include stabilizing accessory proteins, a membrane-mimicking medium called the lipidic cubic phase, and new types of amphiphilic detergents. Another boost came from powerful yet highly focused X-ray beam lines. These allow researchers to collect useful data from GPCR crystals, which tend to be smaller than other proteins'.

Nobody has solved another GPCR-G protein structure. "We absolutely need more," Kobilka says. The structure represents one snapshot in a dynamic process, he adds.

And drugmakers want to see how these kinds of structures can inspire new medicines that can direct GPCRs to couple to different signaling pathways, adds Fiona Marshall, former chief scientific officer of Heptares Therapeutics, a GPCR-targeted pharmaceutical company.

"I think it's too early to say what this structure means in terms of fundamental approaches to drug discovery," Kobilka says. "We're working hard to get other ones."

I watched "Pulp Fiction" in college, at about the same time I first saw a picture of a GPCR. It was a cartoon. Back then, researchers had no GPCR crystal structures. That so much has changed in my short lifetime reassures me of how much is still left to discover.

That noise you're hearing as you read this article is probably the gods having a jolly old time at my expense. As that great American philosopher and poet Yogi Berra was fond of reminding us, it's tough to make predictions, especially about the future.

Still, that hasn't prevented fortune-tellers and stock analysts from making a comfortable living doing it. And as far as short-term predictions go, it isn't as hard as Berra suggests. To a first approxi­mation, tomorrow is usually a lot like today, so we can start by guessing that X-ray diffraction during the next 5 to 10 years is probably going to resemble its present state, only more so. That means more membrane protein structures, more structures of big assemblies, and more use of structural information in the design of drugs and novel proteins.

It also means an acceleration of the use of X-ray diffraction as a routine tool in nanotechnology, materials science, synthetic chemistry, biochemistry, and cell biology. I always knew that crystallography would become a standard technique in the life sciences as it has been in the chemical sciences; I just never imagined that this would happen during my active scientific lifetime. But many of the people who now determine protein and nucleic acid crystal structures were not formally trained in the nitty-gritty of diffraction physics. To them the technique is largely a black box, made accessible by turnkey software that can handle most routine problems.

I sometimes wonder if the people who created such software realized that they were making themselves, if not obsolete, then at least increasingly irrelevant. A quick glance at any history of small-molecule crystallography would have warned them. There used to be departments of crystallography in a number of American universities; now there are none. Not many chemists would call themselves crystallographers any more. I don't think it will be too long before no one will call himself or herself a structural biologist either.

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If you doubt that, consider how few people today refer to themselves as molecular biologists. What was the intellectual discipline of the 1960s and '70s is now part of the routine tool kit of just about everyone in the life sciences. Many small-molecule crystallographers are service staff in chemistry, materials science, and physics departments. One needs no oracle to predict that this will eventually be the fate of many, if not most, macromolecular crystallographers.

In the short term, then, the trends seem to me to be crystal clear (pun intended). In the long run, however … Well, as John Maynard Keynes said, in the long run, we are all dead. But let's not dwell on that.

What makes long-term predictions so difficult in general is the appearance of disruptive technologies, which almost by definition are unforeseeable. The classic example is the polymerase chain reaction (PCR). Before PCR, it took years to clone a gene, and many attempts failed. The day after PCR, anybody could clone anything.

No subject has seen more disruptive technologies than X-ray diffraction. A partial list would include the Patterson function, isomorphous replacement, and anomalous scattering, which enabled the determination of organic structures; direct (i.e., purely computational) methods of phase determination, which enabled small-molecule crystallography to be almost totally automated; synchrotron radiation and area detectors, which together made it possible to collect data on macromolecular structures in hours instead of months; and automatic interpretation of electron density maps. All these technical advances made it easier for expert crystallographers to tackle bigger and more difficult problems. But they also made it easier for people with little or no formal training in diffraction theory to use the technique in chemistry and biology.

Right now, the next disruptive technology in crystallography looks to be the free-electron laser, which produces beams of X-rays so bright that microcrystals can be used for data collection. In one recent experiment, these microcrystals were actually still inside a bacterial cell when they were irradiated. That's right: the structure of a protein in ordered intracellular aggregates was determined without anyone bothering to isolate or purify the protein away from its environment.

An X-ray beam of such intensity will, of course, destroy any microscopic object it irradiates, but with free-electron lasers the diffraction event is faster than the coulombic explosion, so data can still be obtained. Further increases in brightness may enable scientists to determine the structures of very large single particles, especially highly symmetric ones such as viruses, by measuring the diffraction from the particles themselves. There would no longer be a need to pack many copies of smaller objects into a crystal lattice to sufficiently amplify diffraction intensities.

Crystallography without crystals—it sounds like science fiction, doesn't it? But in a sense it's already here. Single-particle cryoelectron microscopy (cryoEM) is now able to provide structures of macromolecular assemblies to resolutions approaching 3 Å, sufficient to allow the polypeptide chain fold to be traced in a number of cases. This technique, which scientists are rapidly automating, may eventually displace crystallography as the tool of choice in many cell biology studies because it does not require the subject to crystallize and sometimes can even reveal multiple conformational states in a single experiment. If you question whether microscopy really should be considered part of the future of diffraction, let me point out that many of the algorithms and other techniques for turning collections of cryoEM images into three-dimensional structures had their origins in X-ray diffraction.

But the most disruptive technology of all is probably one that has actually been with us since the '50s: the computer. It is already possible to predict with surprising accuracy the folds of many simple monomeric proteins directly from their amino acid sequences—no experimental structure determination required. I personally won't get excited about that until its practitioners can do it for large oligomeric proteins—which includes being able to predict what kind of oligomer any given macromolecule will form—and can also predict how a macromolecule's conformation changes when substrate or another macromolecule binds to it. Clearly we are still a long way from the day when all structures of all molecules will simply be calculated from first principles. But if you seriously think that day will never come, I suggest you think again.

Taken together, these considerations suggest that the most definitive statement we can make about the future of X-ray crystallography is that it has no future—or at least, a limited one—in its present form. That sounds ominous, but actually it is not. It isn't extinction we're forecasting here, it's evolution. Prognosticators gleefully predicted the demise of radio when television came along, but radio didn't die out—it simply evolved into a medium with a different purpose. X-ray diffraction as we know it may become obsolete, perhaps sooner than we think, but it seems likely to me that ideas and methods from it will still exist as parts of many, if not most, of the newer techniques that replace it.

I always smile when atomic force microscopists and others who look "directly" at molecular structures get excited about seeing individual atoms. Crystallographers have been doing that for a hundred years. In the end, most experimental science is just an attempt to overcome the limitations of the human eye. The power and glory of X-ray crystallography was that it was the first technique to show our eyes what the atomic world really looked like—initially for minerals and simple solids; then for small organic substances; and eventually for giant molecules, macromolecular assemblies, and even organelles like the ribosome.

If Shakespeare is right and what's past is prologue, the immediate future will, as I've suggested, prominently feature the extension of that vision to even larger biological machines, with the increasing help of methods that incorporate many of the essentials of diffraction but that involve other kinds of radiation and noncrystalline specimens. Beyond that lies the veiled land of things to come, and your guess is not only as good as mine—it may well be better. The future, as Yogi Berra reminds us, is not what it used to be.