Published 29 June 2010 by Ashutosh Jogalekar
Mountains Beyond Mountains
The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed, the measure of our intellectual maturity is our capacity to feel less and less satisfied with our answers to better problems.- G.W. Allport,Becoming, 1955
Science in the popular mind consists of a series of "Eureka!" moments. Such moments are supposed to suddenly propel scientific fields ahead at accelerating rates. Many anecdotes from scientific history seem to confirm this belief. It all begins with Archimedes jumping out of the bath after discovering the principle of buoyancy. Other examples include the apple falling on Isaac Newton’s head, August Kekule waking up from a dream and realizing the structure of benzene, Enrico Fermi discovering slow neutrons by ‘randomly’ substituting a block of paraffin for a tabletop, Alexander Fleming ‘accidentally’ discovering the action of a famous mold on bacteria, and Werner Heisenberg discovering the awesome structure of the quantum world after an all-night session on the island of Heligoland in the North Sea.
Yet those who actually work in the trenches will have different stories to tell. In fact most of science progresses through the quiet and incremental work of thousands of unsung heroes working diligently in their labs and at their desks. Most of these do not get Nobel Prizes. Of course once in a while an Einstein comes along who suddenly makes sense of all this gathered wisdom, but he undoubtedly stands on the shoulders of these toilers. And in the field of toilers, few are as accomplished masters of the art as x-ray crystallographers. X-ray crystallographers will tell you that ‘Eureka!’ moments are usually not part of their daily work. In fact their field is one of the few fields in science where the general goal may be already known. It may be to crystallize hemoglobin, or chlorophyll, or penicillin. Or it may be to crystallize the ribosome, that fearsomely complicated central unit of genetic replication whose study led to the 2009 Nobel Prize for chemistry. Winning a Nobel Prize should never be your ambition, but if it unfortunately is and you want to maximize your chances, you should probably start working on determing the structure of an important protein right away.
Crystallographers more than almost any other breed of scientists are like mountaineers, and x-ray crystallography probably bears the closest analogy of any scientific field to mountain climbing. Through careful, creative and most of all, ferociously patient and determined attempts, crystallographers scale the peaks of molecular structures that beckon the most intrepid of scientific mountaineers. They can already see the peak, but the devil is in the details, in the detours, crevices, forests and gullies that dot the mountain’s sides. And scaling the peak not only provides an unprecedented sense of satisfaction but reveals spectacular views and unexpected riches that inspire, inform and engage. But hold on! The journey is just beginning, because only by scaling one peak can these intellectual mountaineers view others. Only by looking past one can they get a sense of how vast their cherished molecular landscape is. For crystallographers it’s the journey itself that has provided the greatest rewards, for it enables them to see further than others, to get a glimpse of mountains beyond mountains…
X-ray crystallography goes back to Wilhelm Roentgen’s discovery of x-rays at the turn of the century. The story of how Roentgen viewed the ghostly-looking fingers of his hand is well-known. X-rays provided a window into the human body like no other, and quickly became an essential technique for the diagnosis of disease. But if x-rays provided a bounty of tools for doctors, they turned out to be a veritable Ali Baba’s cave for scientists, full of riches beyond their wildest imagination. All this was because of an extraordinarily fortunate property of x-rays: their wavelengths are comparable to the distances between atoms in crystals. This fact would enable scientists to literally shine them on molecular crystals and take a photo, revealing the locations of atoms within them.
The technique was first developed by the German physicist Max von Laue, a man with rock-solid ethics who later bravely stood up to the Nazis. But it was the father and son team of William and Lawrence Bragg who turned x-rays into a practical tool for investigating molecular structure. Since many substances of chemical and biochemical interest can be crystallized, x-rays turned out to be supremely useful for deciphering the structure of matter, and remain so today. Both Braggs made key contributions to crystallography. While the younger Bragg developed Bragg’s equation, a method of calculating atomic spacings based on wavelength, the older Bragg invented the first working x-ray spectrometer, which would generate a steady stream of radiation that would impinge on the molecular target. Lawrence Bragg remains the youngest Nobel laureate, getting the prize when he was 25.
William Lawrence Bragg (1890-1971)
Using the new technique, structures of several inorganic and organic crystals were determined during the period between the two world wars. Starting with simple substances like sodium chloride, crystallographers moved on to more complex organic substances. There were several pioneers in this field, but it was the British who laid the foundations. John Desmond Bernal, a towering intellect and polymath at Birkbeck College who also happened to be an avowed communist (what English intellectual in the 30s was not?), was instrumental in getting crystallography off the ground. His student Dorothy Hodgkin became a legendary crystallographer and won a Nobel Prize. The two solved many important structures including those of vitamins, steroids, penicillin and the tobacco mosaic virus.
Back in Cambridge, Lawrence Bragg became head of the famed Cavendish Laboratories at Cambridge University. If he wanted to pick a location whose history alone maximized the chances of important discoveries, he could not have picked a better place. In the preceding decades the laboratory had been presided over by the towering presence and booming voice of Ernest Rutherford, under whose tutelage no less than 9 Nobel Prize winners were groomed, an accomplishment which is unlikely to be rivaled. Bragg more than sustained the reputation of the laboratory. Under his guidance or at least tacit endorsement, a group of highly gifted men and women gathered after the war to crack the mysteries of life. Among these were James Watson, Francis Crick, John Kendrew, Max Perutz and John Kendrew, all founding fathers of the field of molecular biology.
Together these talented individuals started a bonafide revolution in science that would literally change the world. The revolutionary impact of Watson and Crick’s discovery of DNA structure enabled by Rosalind Franklin’s x-ray photographs is household knowledge and has been seared into our consciousness until the end of time. The discovery is so well-known that retelling it would add only marginal value. Max Perutz’s name is perhaps not as well-known but it should be, since directly or indirectly, he is the godfather of most crystallographers who have won Nobel Prizes since then.
The humble Austrian
Max Perutz (1914-2002)
Perutz was born in Austria and educated in Vienna. As a graduation gift, his girlfriend gave him a copy of Linus Pauling’s landmark work, “The Nature of the Chemical Bond” (a lesson for the young people at Lindau). This book changed Perutz’s view of biology and firmly grounded it in the rational principles of chemistry. He decided to devote his life to the study of molecular structure, but not before he was interned by the British in Canada during the war for being an ‘enemy alien’. One of the more fanciful things that Perutz did during the war was to study the structure of ice and how such knowledge could be used to build gigantic floating airfields made out of a combination of ice and wood pulp in the middle of the Atlantic. Fortunately, the development of long-range fighters and bombers obviated the need for such grand projects, and Perutz’s time could be more gainfully employed elsewhere. After the war he joined the Cavendish and convinced Bragg to let him work on protein structure. For his project Perutz picked hemoglobin, one of the key proteins in living systems that transports oxygen (his colleague Kendrew cracked myoglobin). This was seen as an almost impossibly ambitious goal at the time since previously solved crystal structures had been limited only to a few dozen atoms. In contrast, hemoglobin contains about 140 amino acid residues. Nobody had tackled such a complex structure before.
Solving protein structures requires the resolution of two overarching technical problems, apart from supreme confidence and unyielding stamina. The first problem is getting good quality crystals, and this is where crystallography is transformed into a fine art. Although school students routinely grow crystals of common salt, nobody knows the conditions of pH, temperature, chemical environment and multiple other factors that would be conducive to the crystallization of a particular protein. At some point growing crystals starts to sound almost like black magic. Tricks that have been attempted include slow growth stretching over months to, hold your breath, scratching beards and allowing the tiny bits of hair in them to seed the growth of the crystals. Sometimes, shouting loudly at your flask can also apparently help. But in the end, crystals which may look stunning under the microscope can end up looking abysmal to an x-ray spectrometer. The crystallization problem haunts protein crystallographers to this day. The second problem which plagues crystallographers is a technical issue called the ‘phase problem’. Although it was relatively easy to determine the intensities of atomic reflections, determining the phases of the reflections was quite complex. A number of clever techniques were devised by many scientists including Perutz. One of the most popular has been to deposit atoms of a heavy metal such as mercury in the crystals and then look at the difference in phases between the substituted and unsubstantiated crystals. In practice, solving a crystal structure involves building a model and then back-calculating the expected diffraction pattern from the model. The model is then refined until the calculated patterns matches the observed one.
But all this was in the future. For now Perutz was dealing with something more basic. For solving hemoglobin, Perutz needed to know about the building blocks of proteins and the bonds between them. Amino acids in proteins bond with others through peptide bonds, and the properties of these bonds were just being revealed. Perutz and Bragg found themselves in competition with Linus Pauling, widely acknowledged as the world’s greatest chemist. Unlike Perutz and Bragg, Pauling had an unparalleled knowledge of chemistry and knew bond distances, angles and crystal structures more intimately than probably anyone else in the world. Through his key contributions to theoretical chemistry, Pauling knew a single piece of information better than Perutz and Bragg- that peptide bonds are planar because of the delocalization of electrons over the nitrogen and oxygen atoms. This planarity provides a defining constraint for protein structure. Lying in bed with the flu, Pauling constructed models of polypeptides and hit upon two central structures, the alpha helix and beta sheet. These structures are the foundational elements of all proteins. When Perutz heard about this groundbreaking discovery, he eagerly fixed a horse hair containing the protein keratin in front of an x-ray spectrometer. Just as Pauling had predicted, Perutz saw the x-ray reflections exactly at the positions dictated by alpha helices. Chagrined, Perutz rushed to Bragg’s office. After hearing about Perutz’s missed opportunity, Bragg blurted out “I wish I’d made you angry earlier!”; if Perutz had been provoked enough about his rivalry with Pauling, maybe he would have performed the experiment earlier. Bragg’s quip later became part of a delightful collection of essays that Perutz penned. As for Pauling, he continued to make contributions to an astonishing variety of fields, protested nuclear testing and won a second Nobel Prize for peace, and toward the end of his life became enamored of large doses of Vitamin C as the cure for almost every malady. However, in his second model-building attempt Pauling was embarrassingly unsuccessful. Partly because he was distracted by other things and partly because he did not have access to Franklin’s high-quality diffraction photographs, Pauling would lose the race to crack the structure of DNA. His place in history as the greatest chemist of the twentieth century would still be secure.
Linus Pauling (1901-1994)
Perutz persevered. It took him 15 years to solve the hemoglobin problem, and in doing so he set the trend for future cystallographers. Perutz’s work laid the foundations for understanding the intricate factors that govern protein structure and folding; the protein folding problem is still one of the greatest unsolved problems in chemistry and biology. Important Nobel-winning protein structures solved since Perutz’s time include the photosynthetic reaction center, the potassium ion channel, the machinery of transcription and ATP synthase. Perutz won the Nobel along with Watson, Crick and Kendrew in 1965. Today the hemoglobin molecule is one of the most well-studied of any protein. Perutz remained a scientist’s scientist till the end of his life and was often at the bench even in old age. When he died he was working on the structure of amyloid proteins which are implicated in Alzheimer’s disease, and had sent in a manuscript just a few days before. This brilliant, modest and unassuming man served as an inspiration for the next generation of crystallographers and also wrote many delightful books detailing his and other scientists’ adventures in science. But Perutz was more than a scientist. In 1962 he established the Laboratory of Molecular Biology (LMB) and served as an administrator for many years. The LMB was to biochemistry what the Cavendish had been to physics. It fostered an environment of unique scientific excellence. In running the institute Perutz gave his scientists a free hand and they had an unusual degree of freedom to explore their own ideas. He used to regularly have lunch even with postdocs in the cafeteria and it was primarily due to him that the LMB became a haven of scientific productivity. The LMB has been associated with several Nobel Prize winning crystallographers over the years. No wonder it has nurtured the work of Venki Ramakrishnan, who was a recipient of the 2009 chemistry Nobel.
The ribosome cryptographers
Ramakrishnan won the prize along with Tom Steitz and Ada Yonath for his work on solving the structure of the ribosome. The ribosome, as is familiar to high school students, is the machine that does the job of ‘translating’ RNA into protein. To appreciate the complexity of the problem, it is worth comparing the structure of the ribosome with that of hemoglobin. Hemoglobin is one protein containing 140 amino acids. In contrast, even the bacterial ribosome consists of two subunits called 30S and 50S. The 30S subunit contains 21 different proteins and the 50S subunit contains 34 different proteins. In addition both subunits contain ribosomal RNA (rRNA) molecules. All in all, the ribosome contains about 8000 amino acids and 100,000 atoms which have to be clearly resolved in a structure determination. The magnitude of the problem is fearsome.
The ribosome: A precisely organized jungle of macromolecular assemblies
Among the three recipients of last year’s Nobel Prize, Ada Yonath stands apart for being the first one to have the audacity to address the issue and being the earliest pioneer. In the 1970s, it must have taken Yonath the same kind of courage to tackle the structure of the ribosome as it took Max Perutz to tackle hemoglobin before. Yonath was probably born to be a crystallographer; as she recounts in her Nobel lecture, she grew her first crystals of the enzyme lysozyme when just in 6th grade. She found a clue to growing ribosome crystals from a rather unusual source that is now in danger of becoming threatened- the polar bear. Polar bears were known to pack their ribosomes closely before going into hibernation. After waking up their ribosomes functioned perfectly, indicating their stability. Another clue came from the exotic bacterium Haloarcula marismotui which lives in unbelievably saline environments like the Dead Sea. Yet another organism was a so-called ‘thermophilic’ bacterium which can survive temperatures that would kill almost everything else on the planet. Obviously ribosomes were stable in all these environments. It seemed almost embarrassing that human beings could not grow them under controlled laboratory conditions.
It was from such a thermophilic bacterium that Yonath grew the first 50S ribosomal crystals in 1980. These were grown not through x-ray diffraction but through electron crystallography, where electrons take the place of x-rays in resolving the structure. Electron crystallography can give us crude maps of structure which have to be judiciously interpreted. In the mid-80s Yonath finally obtained the first x-ray crystals. To resolve these she relied on a synchrotron, which has become the tool of choice for researchers wanting intense x-ray beams. Unfortunately synchrotrons are huge, billion-dollar machines and there are only a few throughout the world. Scientists wanting to use them have to reserve time. Yonath used a large synchrotron in Grenoble, France. In the 1986, Yonath had a real breakthrough when she developed the technique of ‘cryobiocrystallography’, a significant technical advance wherein crystals can be stabilized at low temperatures by immersing them in a hydrocarbon environment in which aqueous solvent does not freeze and damage them. This technique saw unprecedented advances not just for ribosome structure but also for other proteins. With these techniques Yonath was finally able to obtain high-quality diffraction data for the ribosome. One of the first key observations she made was that of an ‘exit tunnel’ through which newly synthesized proteins can exit. She could also study the interaction of these proteins with other proteins called chaperones which stabilize them and help them fold. With this work Yonath was well on her way to a Nobel Prize.
The stage was thus set for her co-recipients Venki Ramakrishnan and Tom Steitz. In the early 90s and the first decade of the twentieth century, these scientists determined the structure of the 30S subunit and further delineated the very complex process of protein synthesis which involves several proteins interacting with each other. Biochemist Harry Noller reconstructed the entire 70S bacterial ribosome. These structures confirmed a long-suspected belief that the ribosome was a ‘ribozyme’ which catalyzed chemical reactions and the scientists determined the reaction center. Ramakrishnan and his group discovered a ‘molecular ruler’ which the 30S subunit uses to build polypeptides and fine-tune the accuracy, reducing the error rate to a remarkably low 1 in 100,000 events. Finally, the three groups determined the location of key antibiotics that bind to the ribosome. This knowledge will be valuable in developing new drugs against tuberculosis and other life-threatening diseases. Throughout the long years, the three laureates, their co-workers and students had to solve many technical problems for growing and stabilizing crystals. Yonath had to develop an entire technique for this. Ramakrishnan had to borrow a novel reagent called osmium hexamine which he came across in a research paper (Lesson to Lindau students: Read the literature!). The mountain-climbing analogy was very well-put by Yonath in her Nobel lecture when she said:
”The way to structure determination was long and demanding, we frequently felt as is we are climbing high mountains, just for discovering that a higher Everest is still in front of us”.
She also acknowledged that there were no ‘Eureka!’ moments, although discovering that her technique could give crystals ‘eternal life’ came close.
So what’s next? It is only fitting that the last half-century of astonishing progress in solving crystal structures has only led to more mountains beyond mountains. A recent article in Nature laid out the long road ahead. There are still many challenges. One of the biggest concerns membrane proteins, especially G-protein Coupled Receptors (GPCRs) which are involved in virtually every important life process and are the targets of 50% of all drugs. GPCRs are embedded in membranes and taking them out of these destroys their integrity. Thus it is extremely difficult to crystallize them and to date only a handful of valuable structures have been deciphered. The ribosome is a huge protein complex, but its structure has only allowed us to dream about even bigger biological assemblies. As pointed out in the article, the Nuclear Pore Complex (NPC), the spliceosome which splices RNA and the epidermal growth factor which is an important regulator of cell-function are all waiting to be conquered. And of course, the ribosome for which the Nobel Prize was awarded was the prokaryotic ribosome. Eukaryotic ribosomes are an order of magnitude more multifactorial and complex. Riches will unfold, and spirits will rise.
One of the essential truths about science is that it’s not about results or papers but about questions and progress. Science can never rest on its laurels, for those laurels only encourage further efforts by revealing an intellectual landscape more exciting than the previous one. Thus, while results and experimental end-points are undoubtedly exhilarating, the eye should always be focused on the peak yet unseen, but just within reach. Science cannot progress unless a deep restlessness to know more permeates its practioners. And most importantly, since results are signposts only on the way to more wonders, the best we can all do is enjoy the journey. As Constantine Cavafy says in one of my favorite poems, even if we have then grown old, it would all have been worth it.
Onwards to Ithaca.
When you set out on your journey to Ithaca,
pray that the road is long,
full of adventure, full of knowledge.
The Lestrygonians and the Cyclops,
the angry Poseidon — do not fear them:
You will never find such as these on your path,
if your thoughts remain lofty, if a fine
emotion touches your spirit and your body.
The Lestrygonians and the Cyclops,
the fierce Poseidon you will never encounter,
if you do not carry them within your soul,
if your soul does not set them up before you.
Pray that the road is long.
That the summer mornings are many, when,
with such pleasure, with such joy
you will enter ports seen for the first time;
stop at Phoenician markets,
and purchase fine merchandise,
mother-of-pearl and coral, amber and ebony,
and sensual perfumes of all kinds,
as many sensual perfumes as you can;
visit many Egyptian cities,
to learn and learn from scholars.
Always keep Ithaca in your mind.
To arrive there is your ultimate goal.
But do not hurry the voyage at all.
It is better to let it last for many years;
and to anchor at the island when you are old,
rich with all you have gained on the way,
not expecting that Ithaca will offer you riches.
Ithaca has given you the beautiful voyage.
Without her you would have never set out on the road.
She has nothing more to give you.
And if you find her poor, Ithaca has not deceived you.
Wise as you have become, with so much experience,
you must already have understood what Ithacas mean.
Constantine P. Cavafy (1911)
Note: Apologies to Tracy Kidder for borrowing a title from his book "Mountains Beyond Mountains" about Dr. Paul Farmer, the inspiring physician who has pioneered disease treatments in poor countries. I could not think of a better title to describe the vision of x-ray crystallographers.