The determination of a novel protein structure by X-ray crystallographic analysis involves the following steps. i) Protein purification and crystallisation of the native protein, ii) measurement of native diffraction data, iii) obtaining heavy atom derivatives, iv) measurement and analysis of derivative data, v) calculation of phases, vi) map interpretation and model building and vii) model refinement.
Crystallisation is often the rate limiting step in protein crystallography. Several methods of crystallisation are now well established but application of these methods is still very much trial and error. Crystallisation of a newly isolated protein can take weeks, months or years.
A prerequisite of the solution of the majority of protein crystal structures is to find an isomorphous heavy atom derivative of the native protein. Isomorphous, in this sense, means that the derivative protein crystal structure should be identical to the native protein structure except for the presence of one or more heavy atoms bound to protein molecules, i.e. the lattice, space group, cell dimensions and position and conformation of the protein molecule within the unit cell should be preserved. The most common method of heavy atom inclusion is to soak the native protein crystals in a solution containing a heavy atom compound. Other more recent methods involve the production of protein with modified amino acid residues, e.g. methionine containing proteins can be engineered where selenium replaces the sulphur of the methionine [50].
Once suitable native and derivative protein crystals become available, X-ray diffraction data are collected. One of several procedures may be adopted for data collection. Most single crystal diffraction data are measured using monochromatic X-rays from either a sealed tube generator, a rotating anode generator or from a synchrotron source. The availability of synchrotron radiation has led to the application of the less widely used Laue method of data collection to protein crystallography. It utilises the non-characteristic polychromatic radiation produced by a synchrotron. A detailed description of the Laue method may be found in [47].
Several data collection geometries may be used along with monochromatic radiation. A conventional diffractometer measures each reflection individually with a scintillation counter. A goniometer rotates the crystal so as to satisfy the Bragg condition for each reflection individually while the detector simultaneously records the diffracted X-ray intensity. Diffractometers are still widely used for small molecule work but have recently been superseded by area detectors which are able to measure equivalent quantities of data in a much shorter time; an important factor when crystal samples are highly sensitive to the dosage of X-rays delivered during the experiment. This is particularly relevant for biological samples.
There are two area detector geometries which are at present widely used - rotation geometry and Weissenberg geometry. In both methods the Bragg condition is satisfied by rotating the sample crystal about a fixed axis. A series of diffraction images are measured whereby each image records all reflections satisfying the Bragg condition as the crystal is rotated through a specified angle . A limited value of is necessary so as to avoid overlap of diffracted reflections on an image.
The essential difference is that a Weissenberg camera couples the sample rotation with a translation of the area detector. This helps avoid the problem of overlapping spots and uses the available space on the area detector more efficiently and can further reduce the overall time for data collection. The use of larger angular rotation ranges for each diffraction image in the Weissenberg geometry implies the accumulation of more background radiation per image, however large sample to detector distances, , are also typically used which reduce the level of recorded background relative to the intensity of diffracted X-rays by a factor of .
In a diffraction experiment one can only measure the intensities and diffraction angles of the diffracted beams. All information about the phases of the diffracted X-rays is lost. This phase information along with the amplitudes of the diffracted X-rays is essential for the solution of crystal structures and must be recovered.
There are four approaches which may be taken in recovering phase information in a diffraction experiment. The heavy atom method makes the assumption that if a significant contribution of the scattering from a structure is made by a heavy atom then the phases of the diffracted X-rays will be close to those phases which would be observed were only the heavy atoms present. The problem is thus reduced to finding the positions of the heavy atoms within the structure. This approach is in general not applicable to proteins since the heavy atom contribution to scatteing is small with respect to the protein. The direct methods approach can make estimates about the reflection phases using assumptions about the internal structure of the crystal. Direct methods are routinely used for the solution of small structures and have only recently been applied to the solution of small proteins containing about 50 amino acid residues [102].
If a related or similar protein structure is already known then the method of Molecular Replacement (MR) [98] may be used. The idea is to find the rotation and translation which position the model structure in the unit cell so as to give the highest correlation between experimental diffraction measurements and those calculated from the model. This method relies on the existence of a known related structure and is therefore likely to become more and more applicable as the number of solved protein structures increases in the future.
The third and most prominent of the solutions to the phase problem in macromolecular crystallography is isomorphous replacement and related methods. In these methods phase information is retrieved by making isomorphous structural modifications to the native protein, usually by including a heavy atom or changing the scattering strength of a heavy atom already present and then measuring the diffraction amplitudes for the native protein and each of the modified cases. If the position of the additional heavy atom or the change in its scattering strength is known then the phase of each diffracted X-ray can be determined by solving a set of simultaneous phase equations. Methods which use such a strategy are Single Isomorphous Replacement (SIR), Multiple Isomorphous Replacement (MIR), Single Isomorphous Replacement with Anomalous Scattering (SIRAS) and within the last 15 years, the Multiple wavelength Anomalous Diffraction method (MAD).
With an experimental set of phases obtained from either direct methods or Isomorphous Replacement related methods one can calculate a 3-dimensional electron density map of the protein structure. This is not always readily interpretable as a single polypeptide chain and methods are usually employed to improve the density map using knowledge about the common characteristics of protein crystals. e.g. they nearly always contain between and solvent, are made of individual amino acid residues with known structures and have predictable secondary structures e.g. -helix or -pleated sheet. Density interpretation and model building have been semi-automated with the recent development of powerful graphical computer hardware and software aimed specifically at macromolecular modelling, e.g. the program O [61]. After and during the main phase of model building, refinement of the model is carried out against the experimentally measured intensities. This stage may include the addition of ordered solvent molecules and if very high resolution X-ray data are available even the addition of hydrogen atoms, although this is rare for macromolecular structures.
I was born in Akron, Ohio on June 6, 1943, one year to the day before D-Day, the allied invasion at Normandy. The youngest of four children, I was brought up in a wonderfully stable, loving family of strong Midwestern values. When I was three my family moved to Kansas City, Missouri where we lived in a beautiful large home in a lovely upper-middle class neighborhood. I grew up there (at least to the extent one can be considered to be grown up on leaving for college at age 18) and was convinced that Kansas City, Missouri was the exact center of the known universe.
My mother, Esther Virginia Rhoads, was the third of six children of Charlotte Kraft and Errett Stanley Rhoads, a wealthy manufacturer of furniture in the Kansas City area. She liked the unusual name Errett so much that she gave it to me as my middle name. She picked the name Richard after the crusading English king (the Lion-Hearted), but being a good American and suitably suspicious of royalty, she was fond of calling me "Mr. President" instead. She had big plans for me, and loved me beyond all reason.
My father, Frank Dudley Smalley, Jr., was the second of four children born to Mary Rice Burkholder and Frank Dudley Smalley (Sr.), a railroad mail clerk in Kansas City. Although my father went by the name of June (short for Junior), he never quite forgave his father for not having given him a name of his own, and for not having aspired to more in life. My father started work as a carpenter, and then as a printer's devil, working for the local newspaper, The Kansas City Star, and later for a farm implement trade journal, Implement and Tractor. By the time he retired in 1963 he had long since risen to be CEO of this company, and a group of several others that published trade journals in the booming agriculture industry throughout the Western Hemisphere. He was incredibly industrious, talented, and fascinated with both business and technology. He had a wonderfully analytic mind, and loved argument, open discussion, and homespun philosophy. During the depression in the early 1930's he married my mother (who fell in love with his blue eyes) and was promptly laid off from work. The story of his career is one of total dedication to both his work and his family, a dedication that held steady through a series of tribulatons, many of which I am only now beginning to appreciate. He loved me too, but he could see himself in me, and knew my failings through and through. Until late in life I was never quite good enough for my father, and I suppose that is part of what drives me even now, well after his death in 1992.
My interest in Science had many roots. Some came from my motner as she finished her B.A. Degree studies in college while I was in my early teens. She fell in love with science, particularly as a result of classes on the Foundations of Physical Science taught by a magnificent mathematics professor at the University of Kansas City, Dr. Norman N. Royall, Jr. I was infected by this professor second hand, through hundreds of hours of conversations at my mother's knees. It was from my mother that I first learned of Archimedes, Leonardo da Vinci, Galileo, Kepler, Newton, and Darwin. We spent hours together collecting single-celled organisms from a local pond and watching them with a microscope she had received as a gift from my father. Mostly we talked and read together. From her I learned the wonder of ideas and the beauty of Nature (and music, painting, sculpture, and architecture). From my father I learned to build things, to take them apart, and to fix mechanical and electrical equipment in general. I spent vast hours in a woodworking shop he maintained in the basement of our house, building gadgets, working both with my father and alone, often late into the night. My mother taught me mechanical drawing so that I could be more systematic in my design work, and I continued in drafting classes throughout my 4 years in high school. This play with building, fixing, and designing was my favorite activity throughout my childhood, and was a wonderful preparation for my later career as an experimentalist working on the frontiers of chemistry and physics.
The principal impetus for my entering a career in science, however, was the successful launching of Sputnik in 1957, and the then current belief that science and technology was going to be where the action was in the coming decades. While I had been a rather erratic student for many years, I suddenly became very serious with my education at the beginning of my junior year in the fall of 1959. I set up a private study in the partly furnished, unheated attic of our home, and began to spend long hours in solitude studying and reading (and smoking cigarettes). This happened to be the year when I began to study chemistry for the first time. Luckily, these years were some of the best ever for the public school system in Kansas City, and my local high school, Southwest High, was one of the most effective anywhere in the US as measured by scores on standard achievement tests, and the fraction of students going on to college. My teacher, Victor E. Gustafson, was a great inspiration. He had just begun to teach the preceding year, and was full of love for his subject and for teaching, and had an as yet unblunted ambition to reach even the slowest of students. In addition, this was the first class I had ever taken with my sister, Linda, who was a year older than I, and was a far better student than I had ever been. The result was that by the end of the year, my sister and I finished with the top two grades in the class. We hardly ever missed a question on an exam. It was an exhilarating experience for me, and still ranks as the single most important turning point in my life, even from my current perspective of nearly four decades later. It was the proof of an existence theorem. After my junior year, I knew I could be successful at science. The next year I did equally well in physics with a wonderful professor, J.C. Edwards, but my soul had already been imprinted by my exposure to chemistry the year before.
My mother's youngest sibling, Dr. Sara Jane Rhoads, was one of the first women in the United States to ever reach the rank of full Professor of Chemistry. After earning her Ph.D. in 1949 with William von Eggers Doering, who was then at Columbia University, she devoted her life to teaching and research in the Department of Chemistry of the University of Wyoming. She received the Garvan Medal of the American Chemical Society in 1982 for her contributions to physical organic chemistry, particularly in the study of the Cope and Claisen rearrangements. She was the only scientist in our extended family and was one of the brightest and, in general, one of the most impressive human beings I have ever met. She was my hero. I used to call her, lovingly, "The Colossus of Rhoads". Her example was a major factor that led me to go into chemistry, rather than physics or engineering. One of the most enjoyable memories of my early life was the summer (1961) I spent working in her organic chemistry laboratory at the University of Wyoming. It was at her suggestion that I decided to attend Hope College that fall in Holland, Michigan. Hope had then (and still has now) one of the finest undergraduate programs in chemistry in the United States.
At Hope College I spent two years in fruitful study, but decided to transfer to the University of Michigan in Ann Arborafter my favorite professor, Dr. J. Harvey Kleinheksel, died of a heart attack, and the organic chemistry professor with whom I had hoped to do research, Dr. Gerrit Van Zyl, announced his retirement. While the next two years in Ann Arbor were successful, I had become so entangled in a stormy love affair with a lovely girl back at Hope College, that I was not able to concentrate as much on science as I should have. I did, however, learn a lot. Most of all I learned from my fellow students, and particularly from John Seely Brown, a graduate student in mathematics who lived in an apartment down the hall in a small house off campus (he is currently Director of Xerox's Palo Alto Research Center, PARC). John displayed an audacity of thought and intellectual ambition that I have rarely seen in any individual. My fellow housemates and I were infected with the notion that we could master any subject, and at times we did manage to at least feel that we got close.
By the time of my graduation in 1965, the job market for scientists in the United States was at an all-time high, and even chemistry graduates with just a BS degree were in great demand. Rather than proceeding directly to graduate school, I decided to take a job in the chemical industry in order to buy a bit of time to see what I really wanted to do in science, and to live a little in the "real" world. It turned out to be a terrific decision.
In the fall of 1965 I began work full time in Woodbury, New Jersey at a large polypropylene manufacturing plant owned by the Shell Chemical Company. I began as a chemist working in the quality control laboratory for the plant, a 24 hour a day operation that in the mid 60's was quite a wonderland of high technology. My first boss was a chemist named Donald S. Brath. He taught his young professionals that "chemists can do anything", and the time I worked under him was a wonderfully broadening experience. I was teamed up with chemical engineers at the plant to study problems with the quality of the polymer product. The Ziegler-Natta catalyst system then in use by Shell to produce isotactic polypropylene was no where near as efficient as those currently in use, and the level of inorganics remaining in the polymer was high. Much of what we were concerned with in those days revolved around this problem of high "ash" content and how it affected the downstream applications. These were fascinating days, involving huge volumes of material, serious real-world problems, with large financial consequences. I loved it.
After two years I moved up to the Plastics Technical Center at the same site in Woodbury, and devoted myself to developing analytical methods for various aspects of polyolefins, and of the materials involved in their manufacture, modification, and processing. Although I found my work at Shell highly enjoyable, I realized it was time to get on to graduate school, so I began to study seriously and to send out applications. At the time I was most interested in quantum chemistry, and received several offers for graduate assistantships in excellent schools. I was close to accepting an offer from the Theoretical Chemistry Institute at the University of Wisconsin when the automatic graduate student deferments from the Draft into the US military were eliminated. This was in early 1968, during a major buildup phase in the Vietnam War, and I decided it would be more prudent to remain at Shell for a while since my industrial deferment was still in effect.
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