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Seeing and Knowing in Structural Biology

Open AccessPublished:September 11, 2007DOI:https://doi.org/10.1074/jbc.X700001200
      Chance often determines how a young person finds her calling. In 1949, I was majoring in Biology and Physics at Bryn Mawr College. I took a summer job (as usual) to earn money, this time waitressing at a resort hotel in the Pocono Mountains. We were given uniforms, including hairnets, and were told to prepare for training. The training turned out to be daily practice in serving the older, permanent waitresses, who did not tip and did not like Jews. After a few weeks, I was finally allowed to work in the main dining room. I served my first breakfast to a family of five, my hand shaking as I lowered their juice glasses to the table, and I received my first tip, which was negligible. Feeling hopeless, I telephoned a friend from Bryn Mawr who was spending the summer in Woods Hole, MA, where she worked in a place called the Marine Biological Laboratory (MBL). She urged me to join her there and promised to help me find a job for the rest of the summer. That job gave direction to my scientific life.
      I worked happily in the kitchen of the MBL, tyrannized by the beloved Miss Bell, who treated everyone alike. In my free time, I dissected squid axons (badly) for Otto Schmitt and attended all the lectures I could find. One of the lecturers was a brilliant English mathematician and crystallographer by the name of Dorothy Wrinch. She presented her views (later disproved) on the atomic structures of proteins, with strikingly beautiful slides. I understood little of what she said that summer, but what I saw in her pictures persuaded me to work on the structure of proteins.
      Among the scientists I met at the MBL was young Shinya Inoué, who was then a graduate student at Princeton. He was working on an early version of his now famous polarizing microscope. In a small, darkened room, he showed me a living, unstained egg from Chaetopterus (a marine worm) in polarized light. The spindle fibers were brilliantly clear, and one could detect the “fibrils” (now known to be microtubules) of which they were composed. He then put a Petri dish with the eggs on some crushed ice, and when I looked again, the spindle had disappeared. The mysterious fibrils had depolymerized at low temperature; and this effect was rapidly reversed upon raising the temperature. These discoveries of Shinya showed me that proteins have curious properties, which we, in the late 1940s, were only beginning to understand.
      There it was: I needed to see as much as I could of these marvelous materials. And I needed to comprehend what their images could reveal. Seeing and knowing about protein structures became the main goals of my professional life. I chose to go to graduate school at the Massachusetts Institute of Technology (MIT), where they had a biophysics program with a specialty called “Ultrastructure.” It was a joy to have started graduate work in 1950 when virtually nothing was known about the structure of biological molecules. By the time I graduated in 1954, the α-helix had been proposed by Pauling at Cal Tech, and the DNA double helix deciphered by Watson and Crick at Cambridge and Franklin at King's College, London. The fields that we now call structural biology and molecular biology were emerging (
      • Kendrew J.C.
      How molecular biology started.
      ).

      MIT

      At MIT, under the mentorship of Richard Bear, a kind, modest, profoundly intelligent man and a pioneer in the x-ray diffraction of fibrous proteins, I tackled two problems for my graduate thesis. Both taught me the same lesson. One goal was to determine the structure of the fibrous protein collagen from its rather meager x-ray fiber diagram. Here, thanks to helical diffraction theory, developed by Cochran, Crick, and Vand in 1951 (
      • Cochran W.
      • Crick F.H.C.
      • Vand C.
      The structure of synthetic polypeptides. I. The transform of atoms on a helix.
      ), I knew that I was dealing with a helix and could define its parameters (
      • Cohen C.
      • Bear R.S.
      Helical polypeptide chain configuration in collagen.
      ). But I encountered ambiguities for the exact run of the polypeptide chains and could not resolve them by model building. (I might add that I had to go to the Harvard Botanical Library to examine the old papers on phyllotaxis (the study of the helical arrangement of leaves on plants) to categorize these ambiguities (
      • Cohen C.
      ).) The covalent connectivity of the helices was discovered soon afterward by the insightful work of Ramachandran, followed by that of Rich and Crick.
      I also tackled the problem of interpreting the optical rotatory properties of proteins, including collagen, to try to deduce the sense of twist of the helices they contained (
      • Cohen C.
      Optical rotation and polypeptide chain configuration in proteins.
      ,
      • Cohen C.
      Optical rotation and helical polypeptide chain configuration in collagen and gelatin.
      ). The effects of a helical conformation on polypeptide chain conformation had not previously been examined. Here, the collaboration of a beloved classmate, Paul Gallop, was essential: he prepared the proteins, and I carried out the polarimetry studies. This effort was confounded at that time, however, because we did not know (and even Pauling did not know) whether α-helices were right- or left-handed. Or could they be both? So, early in my work, I encountered the ambiguity involved in interpreting images: seeing an image, or conceiving of one in the mind's eye, often leads to an enigma. In the solution of that enigma, “seeing” can become “knowing.”
      The word “enigma,” deriving from the Greek, means “to speak in riddles,” and the most famous riddle maker is, of course, the Delphic oracle from antiquity. Sitting on a tripod and speaking in a trance, she gave counsel in her so-called “maniacal chatter.” Rightly or wrongly, supplicants interpreted her riddles, which led them to their (sometimes dire) fate. Fortunately, today, in Structural Biology, our misinterpretations are usually not fatal, only embarrassing. In these reflections, I want to show, from my own early experiences and those of others, some of the strategies used in solving scientific riddles.
      When I was at MIT, my early lessons in interpreting images were reinforced by the studies of Hugh Huxley (from Cambridge University) and Jean Hanson (from King's College, London), who were there as postdoctoral fellows. Hugh was expert in the x-ray diffraction of muscle and was learning electron microscopy, as was Jean, who had previously worked in light microscopy. They collaborated in attempting to decipher the relationship between muscle structure and contraction. By 1942, Albert Szent-Györgyi, the Hungarian biochemist who had won a Nobel Prize for his discovery of vitamin C, discovered that two different proteins, myosin and actin, together are essential for contraction. Nevertheless, he and most others believed that there was only one continuous set of “actomyosin” filaments running through a myofibril in muscle and that their internal folding produced shortening. This notion was partly due to the fact that rather thick sections of muscle were being used in electron microscopy at the time. By 1953, however, at MIT, Hugh was obtaining very thin cross-sections of muscle from which he could distinguish two sets of filaments in the region of the so-called “A band” (
      • Huxley H.E.
      Electron microscope studies of the organization of the filaments in striated muscle.
      ). I remember carefully following Hugh's and Jean's studies and seeing how they combined complex and ambiguous x-ray diffraction, light microscope, and electron microscope (EM) images to arrive at a rather radical new mechanism for muscle contraction: the sliding-filament theory (
      • Huxley H.E.
      • Hanson J.
      Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation.
      ,
      • Hanson J.
      • Huxley H.E.
      The structural basis of contraction in striated muscle.
      ). In this theory, they pictured how the myosin molecules were, in effect, pulling the actin filaments into the thick filament lattice to produce shortening.
      A crucial point here is the nature of the evidence. Hugh's and Jean's ideas were accepted only after intense controversy and a variety of additional experimental results (
      • Huxley H.E.
      The double array of filaments in cross-striated muscle.
      ). I remember being struck by the painstaking approach they took; they carried out a number of experiments designed to obtain more and more conclusive evidence. But these same experiments could refute their theory if the results contradicted their hypothesis. In fact, trying to disprove one's own ideas is a common strategy for how to do good science! I should add that, not surprisingly, it was when I was in graduate school that I was converted to the muscle proteins.

      King's College and Rosalind Franklin

      After I took my degree in 1954, I joined Jean Hanson as a postdoctoral fellow at King's College, and the problem I tackled there was the x-ray structure of actin filaments. A technician of Jean's prepared the material, using a method that had recently been developed by Albert Szent-Györgyi's young cousin, Andrew. (He and his wife, Eva, were then working in Albert's laboratory at the MBL in Woods Hole.) The material was very viscous, and I was able to pull highly oriented fibers by dipping forceps into the gel and spreading the tips. The x-ray work was carried out in a room close by, and I used Rosalind Franklin's microcamera to obtain rather good fiber diagrams. Franklin had been at King's until a year before I arrived.
      At that time, I knew almost nothing of the struggle over the structure of DNA that had so recently taken place between King's and Cambridge. Maurice Wilkins (called “Uncle” for his slow seriousness) was at King's, and he and I became quite friendly. In fact, the whole group of young scientists in the laboratory, including Pauline Cowan (later Harrison) and Stewart McGavin, was quite matey, and we often repaired to the nearby pub with Maurice on Friday afternoons. Little was said about Rosalind, but I did know that she had joined J. D. Bernal's group at Birkbeck College, so one day, I arranged to meet her. I duly climbed up to Rosalind's attic office/laboratory at 21 Torrington Square. (I recollect encountering her collaborator, Aaron Klug, on my way up.) She was in a very small room with a desk and chair; on a table behind her were many thin, upright capillary tubes. These contained the oriented samples of tobacco mosaic virus (TMV), her current focus. Rosalind was very gracious, and as she shook my hand, she said diffidently, “You know, I am not really a crystallographer.” I responded that I was not one either and that I just knew a bit about helical diffraction. She showed me her latest x-ray photographs of TMV, which were immensely detailed and very beautiful. And then I made one of my most reckless pronouncements: “You will never solve this structure,” I blurted out. Rather than saluting the perfection of her photographs, I was thinking of how hard it would be to disentangle overlapping Bessel functions. She was very quiet for a moment, and then we went down to a luncheon with several people, including the illustrious Bernal (called “Sage”), whom I met for the first time. He was told that my work was related to muscle contraction, so he proceeded to launch into some theoretical arguments on the subject. And then I behaved badly again; while he was holding forth, I turned to Rosalind to ask, in a whisper, how she could have left Paris for London and had she read Proust? No response. We then parted. She did not entertain me again.
      Some months later, I phoned and said, “Rosalind, I have some really nice fibers of actin, but I need a better camera.” She replied, “I am very sorry, Carolyn, but my camera is tied up all the time.” I understood her message. And within four years, she had disproved my prediction about TMV. Among her contributions, she first identified the location of the RNA (
      • Franklin R.E.
      Location of the ribonucleic acid in the tobacco mosaic virus particle.
      ); then, in collaboration with Ken Holmes, she determined the symmetry of the helical arrangement of the subunits (
      • Franklin R.E.
      • Holmes K.C.
      Tobacco Mosaic Virus: Application of the method of isomorphous replacement to the determination of the helical parameters and radial density distribution.
      ). After her death, Ken, Aaron Klug, and Don Caspar carried this work forward with great distinction.

      MIT, Again, and Andrew Szent-Györgyi

      I spent only nine months of my fellowship in London, with side trips to Paris and Florence and some trips to Cambridge to consult with Crick about α-helices and to take some actin X-ray diagrams with Hugh. I then returned to MIT and Dick Bear's laboratory, happy to be home. Here, I finished my x-ray analysis of actin and was able to define the basic parameters of the structure, but was it helical or planar? (It was not until 1963, when negative staining for EM had been developed, that Jean Hanson and Jack Lowy finally visualized the topology of the actin helix.) I then began some biophysical studies with Andrew Szent-Györgyi, which I will soon describe. One day, most unexpectedly, I received a phone call from Rosalind; it had been about two years since our last interaction. She said, “Carolyn, I am visiting Andrew Szent-Györgyi in Woods Hole, and he has shown me some of your x-ray photographs. Why don't you come down so that we can talk?” I did not know that Rosalind was mortally ill at the time. (It was not until she returned to England that the cancer was diagnosed.) And, to my shame, I had not yet learned the lesson of forgiveness. So I did not make that simple trip, and I regret my decision to this day.
      When I began to collaborate with Andrew Szent-Györgyi at MIT, as Dick Bear had recommended, we began working on optical rotation and x-ray diffraction of α-fibrous proteins, especially those in muscle. Andrew proved to be a delightful partner; he used to bring the proteins with him from Woods Hole to Cambridge, where we carried out the studies. It then became a ritual for us to have dinner in Chinatown and, if possible, go to the Boston Garden to watch the tennis. We were unscrupulous at MIT and often “borrowed” pipettes from better-equipped laboratories (in particular, that of Jack Buchanan). I also used to commute to Woods Hole regularly for our consultations. As with Paul Gallop, our mutual foibles were an immense source of pleasure, and there was much laughing amid the scientific disasters. We were even mildly amused, at a large meeting, to find our work attributed to someone else and then to read our work rewritten, as if de novo, by an admiring colleague. Apparently, our work was of some interest. What we found, in fact, was that many of the fibrous proteins had domains composed of α-helical coiled coils as well as globular regions, and we could begin to relate their shapes to these features (
      • Cohen C.
      • Szent-Györgyi A.G.
      Optical rotation and helical polypeptide chain configuration in alpha-proteins.
      ,
      • Cohen C.
      • Szent-Györgyi A.G.
      The alpha-class of fibrous muscle proteins.
      ).
      I should add that Andrew was also very patient when, after about a year of work, I abandoned him to act out an early fantasy of mine to go to medical school. Somehow, I convinced Boston University to give me a chance, and then I could work with Andrew only on weekends. Part of his patience was due to the fact that he, himself, had earned a medical degree in Hungary while managing to avoid most clinics. He was pessimistic about my future in this field, and rightly so. After less than a month, I came to my senses and realized (not too late, I hoped, for another student to take my place) that medical school was not for me. I never did regret this (mis)adventure, and over time, I became a scrupulous adviser to my own students about careers in medicine.
      I then resumed full-time laboratory work. And here I solved a small, delightful riddle and learned the important lesson that “Great men sometimes make great mistakes.” (I think that great women, in general, tend to be rather more judicious.) In this case, the great man was Linus Pauling, who certainly was one of my heroes. In 1951, he and Robert Corey had published a prodigious number of papers in Proceedings of the National Academy of Sciences of the United States of America on their truly monumental findings about the basic conformations of polypeptide chains (see especially Refs.
      • Pauling L.
      • Corey R.B.
      • Branson H.
      The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain.
      and
      • Pauling L.
      • Corey R.B.
      The pleated sheet, a new layer configuration of polypeptide chains.
      ; for a recent review, see Ref.
      • Eisenberg D.
      The discovery of the alpha-helix and beta-sheet, the principal structural features of proteins.
      ). But in that same volume, they had published a long paper on “The Structure of Hair, Muscle, and Related Proteins” (
      • Pauling L.
      • Corey R.B.
      The structure of hair, muscle, and related proteins.
      ). Apparently, they believed that the report of Lotmar and Picken, in 1942, of “well crystallized” muscle (following Herzog and Jancke, in 1926) should be taken seriously. For several pages, they analyzed the reflections and speculated on the “muscle” structure. This paper was followed, however, by reports in Nature by Dick Bear and his student Cecily Cannan (
      • Bear R.S.
      • Cannan C.M.M.
      The “Lotmar-Picken” X-ray diffraction diagram of muscle.
      ) and by Hugh Huxley and John Kendrew (
      • Huxley H.E.
      • Kendrew J.C.
      Extractability of the Lotmar-Picken material from dried muscle.
      ) showing that a water-soluble, small, organic molecule was, in fact, the source of the “crystalline muscle” diagram. My contribution, in 1958, was simple: I noticed that most of the muscles that yielded this pattern were derived from mollusks. Seeking advice, I telephoned Betty Twarog, a physiologist friend of mine at the Harvard Biology Laboratories, to ask what substance might be found in high concentrations in molluscan muscle. She quickly named a few compounds, including taurine (an amino acid). I then soaked a washed frog sartorius muscle in a 5% solution of taurine, let it dry, and took the x-ray photograph. There was the Lotmar-Picken diagram! The note I soon published (
      • Cohen C.
      The Lotmar-Picken substance: Taurine.
      ) was just one paragraph in length and had just one figure. Would that more scientific experiments could be so simple and diverting!

      Don Caspar and Tropomyosin

      I first met Don Caspar before I finished my Ph.D. He visited me, rather unexpectedly, in Cambridge, MA, where I was then living. He was a close friend of a classmate of mine at Bryn Mawr, Ethel Stolzenberg (later Tessman). In 1945, I had met Ethel in New York City, when both of us were standing outside Barnard College, waiting for a scholarship interview. She was a Brooklyn original: her father was a revered Yiddish poet; her clarity and humor were incomparable. A year later, we met again at Bryn Mawr, where we were both majoring in Biology. Eventually, she developed the deluded notion that a biophysicist named Don Caspar was destined to become my life partner. When we finally met for the first time in Cambridge, Don was finishing his thesis at Yale, and I would soon be off to London. He said he was working on the small-angle equatorial diffraction of oriented TMV liquid crystals. This did not inspire my optimism, but, fortunately, I had laryngitis at the time and could not comment.
      While I worked at MIT, Don went to Cal Tech and then to Cambridge, UK, for a postdoctoral stint. After he returned to Yale, we began our collaboration in 1957. I had decided to try to solve the structure of the beautiful, highly hydrated tropomyosin crystals. These had been discovered, in the 1940s, by Kenneth Bailey at Cambridge, and I asked Andrew Szent-Györgyi to crystallize some tropomyosin for me. (I should note that some years earlier, when thinking about α-helices, Crick had carried out one of his rare experiments by trying to take an x-ray photograph of a tropomyosin crystal at Cambridge, but succeeded only in burning a large hole through the crystal.) I found that our x-ray equipment at MIT was too primitive to take precession photographs, so I telephoned Don, and he invited me to his laboratory at Yale. In November, I drove down from Boston to New Haven, where Don met me. We managed to get the fragile crystals into a capillary and then threw the windows open in the x-ray laboratory to cool the room while Don's postdoctoral fellow, Bob Langridge, set up the camera.
      That evening, Don put me up, fed me an excellent dinner, and gave me a copy of C. P. Snow's The Search to read. (This novel was based on Bernal's colorful career in the 1930s.) Next morning, we examined the photograph and were astonished: we saw two spikes of strong reflections, forming a dramatic cross. We knew we had an important result, but the photograph did not speak to us until some years later.

      The Scientific Commune at the Jimmy Fund, Boston

      After my post-doctoral years in the Bear laboratory at MIT, Dick was leaving to become a Dean at Iowa State University, and I was beginning to see that it was time to establish my own laboratory. I wanted to find a place where I would be free to carry out research and where no one would tell me what to do, a very privileged place, as I was well aware. (In those days, modest National Institutes of Health research grants were relatively easy to obtain.)
      I had become friends with Betty Geren (later Uzman), a post-MD student of Frank Schmitt at MIT. She had the great distinction of having discovered, by a series of electron microscopic studies, how myelin is formed in the nervous system. Betty had established an electron microscopy laboratory at the fledgling Children's Cancer Research Foundation in Boston (also called the Jimmy Fund), directed by the extraordinary pathologist Sidney Farber. Dr. Farber was a visionary about curing cancer, especially the kinds that afflict children. He was also a realist about money and politics at the Harvard Medical School, where he was one of the first Jews to become a professor. (He sometimes likened the “Quadrangle” at the Medical School to the Roman Arena.) After I set up a small x-ray laboratory for Betty, she convinced Dr. Farber to offer me some space of my own at the Jimmy Fund. (It was no accident, in those days, that many less-than-illustrious places recruited a number of first-rate women.) This seemed to me an excellent opportunity, and I was elated when Don Caspar agreed to abandon the tenure track at Yale to throw in his lot with me in a joint venture. That was the beginning of our happy commune: we shared the expenses of the equipment, and far better, we shared ideas.
      We established our laboratory about 1958, and the place began to flourish. Bob Langridge and Susan Lowey soon joined our commune. Susan, a physical chemist who had been working on myosin in John Edsall's laboratory at Harvard, started as a postdoctoral fellow with me. Together, we tried to envisage a structure for myosin by combining her expertise on the hydrodynamic behavior of myosin and the meromyosins with the information from wide-angle x-ray diffraction and optical rotatory dispersion that Andrew Szent-Györgyi and I had obtained (
      • Cohen C.
      Invited discussion.
      ). We conceived of a model for the molecule that had a “double-stranded α-helical core with globular mass(es) projecting from the rod” (
      • Lowey S.
      • Cohen C.
      Studies on the structure of myosin.
      ). Some five years later, Susan and Henry Slayter visualized myosin in the electron microscope and showed that there are, in fact, two globular heads to the molecule (
      • Slayter H.S.
      • Lowey S.
      Substructure of the myosin molecule as visualized by electron microscopy.
      ). This achievement was among the earlier of Susan's many notable contributions to studies on the biochemical dissection of myosin.
      Ken Holmes came over from England in the fall of 1959 to continue his work on TMV as a postdoctoral fellow with Don Caspar. Trained by Rosalind Franklin, Ken was expert with focusing monochromators, and he was soon helping me take diffraction photographs of Betty Twarog's favorite molluscan muscle, the anterior byssus retractor muscle of Mytilus edulis, which controls the byssus threads of mussels and enables them to cling to rocks. This so-called “catch muscle” had an ideal anatomy for physiologists to relate its mechanical and electrical properties. It also turned out to be an ideal specimen for displaying the diffraction pattern from α-helical coiled coils because of the large amount of the α-helical coiled-coil protein paramyosin in the core of the thick filaments. (This is another excellent example of how Nature always provides the “right” creature to answer any question a biologist might ask, analogous to the concept of “privileged materials” described by Louis Pasteur in connection with crystals of the tartrates that he exploited to discover the world of stereochemistry.) The intact cylindrical muscle could readily be pulled into a quartz capillary and yielded the first x-ray diagram of hydrated, well oriented α-helical coiled coils. This fiber diagram spoke plainly to Ken and me, and we were able to establish the two-chain structure of the molecules, which had been the subject of controversy until that time (
      • Cohen C.
      • Holmes K.C.
      X-ray diffraction evidence for alphahelical coiled coils in native muscle.
      ). But the patterns were not good enough to distinguish whether the chains were parallel or antiparallel, and we blundered by choosing the latter. It was only when Don and I finally made progress with tropomyosin (described below) that the answer became plain. In fact, it took until 1991 for the atomic details of an α-helical coiled coil to be established with the crystal structure of the leucine zipper GCN4 (
      • O'Shea E.K.
      • Klemm J.D.
      • Kim P.S.
      • Alber T.
      X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil.
      ). I should add that Ken (I trust because of our work together) switched his focus from virus structure to the muscle proteins, a field in which he has become a renowned expert.
      Another general point we made, based on the coiled-coil structure, was that the α-helix in any protein in aqueous solution acquires increased stabilization by intramolecular side chain interactions. In all proteins, hydrophobic bonding is a primary driving force for tertiary fold formation in aqueous solution. But the interactions of α-helices in globular proteins are complex: they do not display a simple, systematic scheme, as is found in coiled coils. Nevertheless, isolated regions of secondary structure, whatever their conformation, simply are not stable. When one does find such regions, they are often hinges or points of lability. This is a key message of the coiled coil.
      As I have described elsewhere (
      • Cohen C.
      Good times with Don Caspar.
      ), Don Caspar is a great explainer. He and Aaron Klug had been trying to explain the construction of icosahedral viruses to each other for a number of years. Following Rosalind Franklin's death in the spring of 1958, they worked together at the Medical Research Council (MRC) in Cambridge, UK, to write the paper on virus structures that she was to have presented at a symposium that winter. Later, and in part inspired by the design of geodesic domes by the architect Buckminster Fuller (another explainer), they made a major breakthrough. They accounted, with great precision, for the hitherto puzzling numerology found in the so-called “morphological” units that were seen, by EM, clustered on the surface of icosahedral viruses. They formulated, as well, the concept of self-assembly of biological structures (
      • Caspar D.L.D.
      • Klug A.
      Physical principles in the construction of regular viruses.
      ) by showing how the design of the assembly is built into the bonding pattern of the subunits. Aaron came to the Jimmy Fund in 1962 to write this paper with Don, and I remember how assiduously he quizzed me about coiled coils. Then he and Don went to the Cold Spring Harbor meeting, where they announced their solution to the virus construction problem. That certainly was a high point of Don's (and the laboratory's) accomplishments.
      At this time, Don was still working with Ken Holmes to interpret the curious x-ray pattern of the Dahlemense strain of TMV they had obtained in 1960. On the basis of my experience with diffraction from coiled coils, I was able to point out to them that a periodic deformation of the TMV helix could produce the diffraction effects seen on the diagram (
      • Caspar D.L.D.
      • Holmes K.C.
      Structure of Dahlemense strain of tobacco mosaic virus: a periodically deformed helix.
      ).
      The CIBA Foundation meeting that was held in London in 1965, “Principles of Biomolecular Organization,” was an exciting reunion of many of the key workers in the major fields that were then under study. Don Caspar had arranged the meeting, and Francis Crick was the chairman. In the Preface to the proceedings of the meeting, the Foundation praised Crick for his “vigorous” leadership of the discussions. I can certainly attest to how vigorously he tried to bully me in my presentation about fibrous proteins (
      • Cohen C.
      Discussion of the design and function of fibrous proteins.
      ), but I held my own and managed, I believe, to teach him something. One of my messages was that there are three levels of organization in fibrous proteins: the monomer, the aggregate, and the covalently cross-linked polymer. In the case of structural proteins, such as collagen and fibrinogen, assembly proceeds to the covalently bonded polymeric state. By contrast, proteins involved in motility lack cross-linking, a feature that allows them to be dynamic. Many other topics, such as self-assembly, were discussed at the CIBA meeting, which was a marvelous survey of the great progress that was being made in biomolecular structure at the time.
      Meanwhile, Don and I persisted in our efforts on tropomyosin. Bill Longley and I were also carrying out various experiments on this protein. Knowing the importance of Ca2+ in muscle contraction, I asked my technician to add ∼10–5 m CaCl2 to a solution of tropomyosin because this level of Ca2+ is required in regulation. The next morning, we found that the protein had precipitated. In the light microscope, we saw birefringent needles and, by electron microscopy, beautifully ordered paracrystals with a striking 400 Å periodicity. It pleased me, of course, to imagine that the atmosphere in our laboratory was truly a bit magical, but what had really occurred? It turned out that my generous assistant had added ∼1000 times the concentration of Ca2+ ions that I had suggested. By this happy accident, we discovered what others have called “Cohen-Longley paracrystals” (
      • Cohen C.
      • Longley W.
      Tropomyosin paracrystals formed by divalent cations.
      ). This is the only scientific discovery to bear my name.
      Don and I were dismayed to find that the crystallographic patterns of tropomyosin still did not speak to us. Then Hugh Huxley (first at University College, London, and later at the MRC in Cambridge), using negative staining in the EM (in which protein molecules are outlined against a dense heavy metal background) on a variety of muscle proteins, discovered that fragments of Bailey's crystals displayed a kite-shaped mesh of cross-connected filaments (
      • Huxley H.E.
      Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle.
      ). One day, I was discussing the x-ray patterns with Don, and I said to him with some frustration, “Look, Don, we have to connect these EM images with our x-ray diagrams!” He reached into the wastepaper basket and pulled out a recent x-ray photograph. We stared at it, and then, the photograph spoke (in Don's voice) at last! It told us that we were looking at the same view of the crystal as in Huxley's electron micrographs. Using optical diffraction, we could show how the two sets of strands in the mesh, seen in the EM, could produce the dramatic cross of reflection that was seen by x-ray diffraction. The x-ray patterns also told us that the tropomyosin molecules are polar, i.e. one end of the molecule is different from the other; thus, the two chains are parallel, so the molecules have a top and a bottom.
      Galvanized by these results, we wrote a series of papers on the subject, which we subsequently presented at a variety of meetings (
      • Caspar D.L.D.
      • Cohen C.
      • Longley W.
      Tropomyosin: crystal structure, polymorphism and molecular interactions.
      ,
      • Caspar D.L.D.
      • Cohen C.
      Polymorphism of tropomyosin and a view of protein function.
      ,
      • Cohen C.
      • Caspar D.L.D.
      • Parry D.A.D.
      • Lucas R.M.
      Tropomyosin crystal dynamics.
      ,
      • Cohen C.
      • Caspar D.L.D.
      • Johnson J.P.
      • Nauss K.
      • Margossian S.S.
      • Parry D.A.D.
      Tropomyosin-troponin assembly.
      ). The opening sentence of one of those papers was typical of our playfulness: “There are few protein crystals, seriously studied, which give poorer x-ray diffraction patterns than tropomyosin” (
      • Cohen C.
      • Caspar D.L.D.
      • Parry D.A.D.
      • Lucas R.M.
      Tropomyosin crystal dynamics.
      ). I believe that it was through this work that David Parry, then a postdoctoral fellow in our laboratory, was first introduced to tropomyosin and actin, fields in which he has since made many notable contributions. (He has also become an essential, albeit distant, collaborator and good friend.) This was the first protein crystal whose structure was determined, to a resolution of 20 Å, from electron micrographs. One of the obvious findings at the time was that the bending of the tropomyosin filaments in the lattice (produced by the cross-connections) into a coiled-coil coil is related to the bending of tropomyosin as it winds around the actin filament in muscle.
      Another concept that we recognized from the crystals and paracrystalline forms of tropomyosin was that the molecules have a conserved and invariant head-to-tail connection, whereas other linkages in the lattice, such as crossover points, are variable. A similar concept applies to many other molecular machines in which the stability of the structure is provided by conserved linkages, within or between molecules, while its functions (in different states) are carried out, using variable linkages (
      • Caspar D.L.D.
      • Cohen C.
      Polymorphism of tropomyosin and a view of protein function.
      ).

      More About Self-assembly

      A key corollary of the concept of self-assembly is that structures formed in vitro are often similar to those found in vivo. As cited above, Hugh Huxley showed this clearly, especially for myosin, in his EM study of the comparison between “native” and “synthetic” protein filaments from vertebrate striated muscle (
      • Huxley H.E.
      Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle.
      ). In the late 1960s, Andrew Szent-Györgyi and I began to focus on a similar comparison of paramyosin found in the cores of the thick filaments of molluscan “catch” muscles and its in vitro assembly. Negative staining of these cores showed a striking “checkerboard” array of electron-dense nodes in the EM. One of the nicest discoveries we made was that we could reproduce, in vitro, this native packing of the paramyosin molecules. By comparing a variety of the in vitro forms, we could easily account for this checkerboard array by a specific shifting of polar subfilaments. The molecules in the subfilaments did not bond end to end, but had gaps and overlaps in the assembly (
      • Cohen C.
      • Szent-Györgyi A.G.
      • Kendrick-Jones J.
      Paramyosin and the filaments of molluscan “catch” muscles. I. Paramyosin structure and assembly.
      ); these gaps were seen as the dark regions where stain accumulated. (This arrangement also revealed the length of the paramyosin molecule.) A related gap-overlap assembly had first been shown to exist for collagen fibers by EM studies (Ref.
      • Hodge A.J.
      Structure of collagen at the electron microscopic level.
      ; see also Ref.
      • Cohen C.
      Discussion of the design and function of fibrous proteins.
      ).
      Some years later, after collagen had been sequenced, the physical basis for its assembly was shown to be due to both long-range repeats of complementary charge interactions and apolar interactions (
      • Hulmes D.J.S.
      • Miller A.
      • Parry D.A.D.
      • Pies K.A.
      • Woodhead-Galloway J.
      Analysis of the primary structure of collagen for the origins of molecular packing.
      ). Later, still, with the sequencing of myosin and paramyosin, long-range repeats of complementary charge interactions were found to be the basis for intermolecular recognition and to account, as well, for the remarkable periodicities seen in the native protein filaments (see, for example, Ref.
      • McLachlan A.D.
      Structural implications of the myosin amino acid sequence.
      ; for summary with references, see Ref.
      • Cohen C.
      Why fibrous proteins are romantic.
      ). In fact, analysis of these in vitro forms reveals precise features of molecular structure and packing that are far more difficult to analyze in the native filaments. Complementary recognition sites are the key to fibrous protein assembly.
      Here I must add that the physical basis of molecular recognition was plain to Linus Pauling in the 1930s and 1940s, before any large structures had been solved. He suggested that “biological specificity results from the interaction of complementary molecular structures,” held together by weak forces (
      • Pauling L.
      The molecular basis of biological specificity.
      ). This molecular matching, he believed, is the real secret of life.

      The Jimmy Fund Laboratory, Revisited

      There were many other gifted young scientists in the laboratory. Lubert Stryer was soon working with me on the small-angle x-ray structure of fibrinogen (another α-fibrous protein). We were able to show, by a simple experiment, that the early notion of Bailey, Astbury, and Rudall in the 1940s had been correct: “Fibrin is no other than an insoluble modification of fibrinogen” (
      • Stryer L.
      • Cohen C.
      • Langridge R.
      Axial period of fibrinogen and fibrin.
      ). Eventually, Lubert went on to greater glory (both in science and in textbook writing), while the fibrinogen-fibrin conversion became a long-term interest of mine.
      Stephen Harrison was also there as Don Caspar's first graduate student. Steve began his heroic study of an icosahedral virus structure, first by constructing a novel focusing x-ray camera suitable for such giant molecule crystallography and then by using patient visual inspection for measuring the many reflections on his x-ray photographs. A decade after he completed his thesis on the low-resolution virus structure, Steve succeeded in determining the first atomic structure of a crystalline virus, which was followed by many other notable achievements.
      I must mention that Don Wiley, whose memory I will always cherish, was also a graduate student of Don Caspar in our laboratory. Eventually, he and Steve Harrison set up their own Structural Biology Laboratory at Harvard College. Young as he was, Don Wiley nevertheless became a kind of adviser to me about the sometimes bewildering ways of professional science.

      A New Beginning

      We were active on a number of fronts in our laboratory at the Jimmy Fund during that time, but it eventually became clear, with Dr. Farber's failing health, that we should seek to move the group, as a group, to another location. Of course, it was not easy then, just as it is not easy now, to secure three tenured appointments, including two for women, in any research university setting. After considering a few possibilities, we found that Brandeis University, in nearby Waltham, was building the Rosenstiel Basic Medical Sciences Research Center. Fortunately for us, Andrew Szent-Györgyi was on the faculty in the Department of Biology there, and Sidney Farber was a University Trustee. We officially took the name “Structural Biology Laboratory” and, in 1972, became the first research group at the Rosenstiel Center. Don joined the Physics Department; Susan, the Biochemistry Department; and I joined Biology (Fig. 1). We brought with us our ingenious machinist, Charles Ingersoll, who, like his father before him, fashioned the innumerable models on which we (especially Don) relied for our daily attempts at seeing and knowing. Within a year, we were joined by David DeRosier, who, with Aaron Klug, had developed three-dimensional reconstruction for electron micrographs and who was initially appointed to the Physics Department. Since then, we have all been working in structural biology, both together and with colleagues, for some 30–35 years. Hugh Huxley joined us in 1987, after his remarkable career at the MRC in Cambridge.
      Figure thumbnail gr1
      FIGURE 1A new beginning: Susan Lowey, Don Caspar, Carolyn Cohen, and David DeRosier (clockwise) around the “Wand of Inquiry,” outside the Rosenstiel Basic Medical Sciences Research Center, Brandeis University, circa 1973.
      When we moved our laboratory from the Jimmy Fund to Brandeis University in 1972, we also began to move, in effect, from fibers to crystals (see Ref.
      • Cohen C.
      Why fibrous proteins are romantic.
      ). Advances in x-ray technology and molecular biology were now available, and it became possible to determine the structures of our molecules at the atomic level. Perhaps it was fitting that the motto of Brandeis is the Hebrew injunction (from Psalm 51) to pursue “Truth, even unto its innermost parts.” We thrived at Brandeis.

      Perspective

      Now, 58 years after my first summer at Woods Hole, I find myself once again in the library of the Marine Biology Laboratory, remembering all the faces, the papers, the amazement, and the joy of these first 2 decades of my life in science. In my mind's eye, I look back on the lessons we learned from our work and the people who were essential to the happiness of our enterprise. From this perspective, I want to make some general remarks about structural biology.
      Many of us have always believed that an understanding of structure leads to an understanding of function, but this path is not always straightforward. As it turns out, general ideas about function are often derived from relatively low-resolution information, such as that obtained from certain forms of light microscopy, electron microscopy, and low-resolution x-ray diffraction. This was certainly true for the sliding-filament theory of muscle contraction, as well as for the solution of the packing in icosahedral virus particles. Atomic structures of the proteins were not necessary for either proposal. Higher resolution data about finer aspects of structure, however, can provide a far more complete picture. For example, only in recent years has information about detailed conformational changes in myosin cross-bridges been obtained from crystals of subfragment 1 (S1), the so-called “head” of myosin. Even in the absence of actin, certain conditions have produced crystals that reveal aspects of the actin-myosin interaction (
      • Coureux P.D.
      • Wells A.L.
      • Menetrey J.
      • Yengo C.M.
      • Morris C.A.
      • Sweeney H.L.
      • Houdusse A.
      A structural state of the myosin V motor without bound nucleotide.
      ,
      • Coureux P.D.
      • Sweeney H.L.
      • Houdusse A.
      Three myosin V structures delineate essential features of chemo-mechanical transduction.
      ,
      • Yang Y.
      • Gourinath S.
      • Kovàcs M.
      • Nitray L.
      • Reutzel R.
      • Himmel D.M.
      • O'Neall-Hennessey E.
      • Reshetnikova L.
      • Szent-Györgyi A.G.
      • Brown J.H.
      • Cohen C.
      Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.
      ). These results can be coordinated with such advances as the visualization of individual motor molecules. The physical basis underlying general theories can emerge, then, from knowledge at a very fine level.
      Critical technological advances continue to be made. Almost the entire human genome had been sequenced by 2001, bringing us to the so-called “post-genomic” era. The speed with which relatively simple structures are now determined by crystallographic and NMR methods is remarkable; a number of so-called “high-throughput” centers are now in operation. Some believe that this new discipline of “proteomics,” or “structural genomics,” will be key to revealing how individual proteins interact with one another and function in the cell (
      • Kim S.H.
      Structural genomics.
      ).
      We certainly are speeding up structure determination, and we are hoping to encompass a far broader range of inquiry. A number of scientists have tackled the riddle of “Whither Structural Biology?” (
      • Harrison S.C.
      Whither structural biology?.
      ,
      • Abad-Zapatero C.
      Notes of a protein crystallographer: quo vadis structural biology?.
      ). Harrison foresees a fusion of “structural molecular biology” and “structural cell biology” into a unified discipline within the decade, which will allow us to understand the cell as never before. But Abad-Zapatero thinks that this goal is insufficient because it is too static. We must also take into account the concept of “dissipative structures.” Most cellular functions, after all, take place far from equilibrium. He dreams of mapping, “in space and time,” the dissipative structures that are the essence of all biological systems (
      • Abad-Zapatero C.
      Notes of a protein crystallographer: quo vadis structural biology?.
      ). I think that both predictions imagine achieving an understanding of cellular processes and, perhaps, whole organisms by a reductionist, bottom-up approach.
      Advances will be rapid, of course: there will be a continuous crossing of boundaries, and labels will have less meaning. A matter of concern, however, is that too much information, without adequate comprehension, may not really clarify general concepts, which are best understood by inductive, top-down, or what may be called “holistic” thinking (see Ref.
      • Gierer A.
      Holistic biology: back on stage?.
      ). In structural biology, deriving general ideas of function from low-resolution information is such a top-down approach; an overwhelming amount of information is not needed. I suggest that, as always, it will be individual creativity, integrated perhaps with some high-throughput enterprises, that will permit our passage from seeing to knowing in understanding the cell (
      • Cohen C.
      Seeing and knowing in structural biology.
      ). Undoubtedly, we will find answers to questions that have not yet been asked.

      Gratitude

      Looking back now, I recognize how privileged I was, early on, to have had so many good collaborators. By “good,” I mean smart, complementary in talents, and, most important, possessing a lively sense of humor. Science, despite some famous aberrations and popular misconceptions, is fundamentally a shared enterprise. Collaborators become friends of a special kind. My first long-term collaborator was the charming Andrew Szent-Györgyi, with whom I continue to work and wrangle every day. Susan Lowey has achieved great distinction as a scientist (and gifted artist). She moved her laboratory to the University of Vermont in 1998, and we remain good friends. Many of the other friends I made in my first years as a scientist were closely connected to Rosalind Franklin; she remains a presence, even now, in all of our lives. Of course, there was Don Caspar. There was also Vittorio Luzzati, whose wit and delight in debate I have treasured since we first met at MIT, around 1958. There were Anne and David Sayre, an immensely intelligent and civilized couple whom I met at Cold Spring Harbor in 1971. They invited some of us at the Cold Spring Harbor meeting to their nearby home to talk about Rosalind for the book that Anne was writing to challenge Jim Watson's best-selling conceit. Anne's excellent book was published in 1975; she died in 1998. David and I still talk together often. Here, I must also mention my admiration for Aaron Klug, Rosalind's collaborator and friend, who has never stopped seeking justice for her memory. I salute him for his scholarly articles in Nature, following Watson's book, and for his Nobel address and Presidential address for the Royal Society (see also Ref.
      • Klug A.
      The discovery of the DNA double helix.
      ).
      I also treasure the memory of the late John Kendrew, a dear friend who was a great scientist, a great leader of scientists, and the first Director of the European Molecular Biology Laboratory. I must acknowledge the steady influence of Alfred Gierer, a gentle person and profound thinker whom I was fortunate to befriend in my graduate school days at MIT. And I wish there were space to cite the many other early investigators whose beautiful work provides the foundation for our present views in structural biology. I would add that my work has been greatly enriched by the exceptional talents and dedication of students and colleagues in my own laboratory.
      I cannot adequately express my gratitude to my parents. My father was a magnetic, outrageous, and brilliant presence in my childhood who died at the age of 39 after a long illness. My mother then began work as a model and salesperson in hat salons in New York City to support my sister and me. My mother's great goodness, courage, and hard work carried us through those years. I dedicate these pages to her memory and to my partner, Barbara, who understands very well the significance of “Remembrance of Things Past.”

      Acknowledgments

      I am pleased to acknowledge the continuous support of the National Institutes of Health for my work, as well as the support of the National Science Foundation and the Muscular Dystrophy Association. I also thank Don Caspar and Daniela Nicastro for comments on this manuscript and Linda Lynch for devotion to the preparation of this manuscript.

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