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 New York City in 1918 into a family that had a number of artistic people among its members. My father's brother and a sister's husband were probably the best known. The latter, Ivan Olinsky, taught for many years at the Art Students' League in New York City. I have been told that my paternal grandfather professionally made artistic decorations in peoples' homes. The propensity for artistic endeavors extended to my generation and beyond.
My mother was an excellent pianist and organist and it was one of her hopes that I would become a professional pianist. As a youth I was entered into "Music Week" competitions in New York City. I had some modest success, but found at an early age that I had no taste for public performance. On the other hand, I was strongly attracted to science as a lifelong career at an early age.
I had the privilege of attending schools in the New York City public school system. Their standards of education, character building and discipline were very high and I, most certainly, benefited from them. They separated out the more advanced students and permitted them to progress at their own pace. In my case, this occasionally led to some curious circumstances. In my senior year in high school (Abraham Lincoln), the girls would join the boys to practice dancing. I was 14 years old at the time and the girls were the usual 17-18 years old. The physical discrepancy between this 14 year old boy and 17-18 year old girls was considerable. Their first reaction was incredulity but after a while they got used to my presence and even danced with me. I took the chemistry and physics courses that were available, both taught by the same man. He recognized my interests and was very encouraging to me.
I enjoyed a number of sports that I participated in at every opportunity, swimming in the ocean nearby, a game called single-wall handball, played with a little hard black ball and well-known mainly in some metropolitan areas, touch football whose rules eliminate the bruises from tackling and ice-skating that was facilitated by the flooding of a huge parking lot by the local fire department.
I entered the City College of New York in 1933 and, at first, found it to be a bit of a struggle. Their academic standards were very high and they had a concentration of the best students in New York City. In addition, I spent three hours a day traveling on the subway system to and from home. This marked the end of piano practicing. City College had no tuition fee. The only financial requirement was one dollar per year for a library card. At the College, there were broad course requirements for all students that ranged through mathematics, the physical sciences, the social sciences and literature. There were even two years of compulsory public speaking courses. I studied, in addition to the requirements, some additional mathematics, some physics, and much chemistry and biology. The year after graduation from City College was spent at Harvard University in the study of biology, for which I received a master's degree, M.A., in 1938.
After a brief hiatus, I went to work with the New York State Health Department in Albany. While there, I had the opportunity to spend some time again at the piano. At the time I was in Albany, the fluoridation of drinking water was getting underway. I developed a procedure for determining the amount of fluorine in water supplies that became a standard method. This was my first modest contribution to science.
It was my intention to save enough money while at the Health Department to return to graduate school. This I did, and I entered the Chemistry Department of the University of Michigan in 1940 where I met my wife, Isabella Lugoski, whom I married in 1942, at an adjoining laboratory desk the first day that I went to physical chemistry class. We were both attracted to physical chemistry and took our degrees with Professor Lawrence O. Brockway whose speciality was the investigation of gas-phase molecular structure by means of electron diffraction. Although my Ph.D. degree was awarded in 1944, I had completed all my work for it during the summer of 1943 and went off to work on the Manhattan Project at the University of Chicago. Isabella joined me on this project a few months later.
In 1944, we returned to the University of Michigan, I went to work on a project of the Naval Research Laboratory and Isabella as an instructor in the Chemistry Department. While at the University of Michigan, I performed some experiments on the structure of monolayers of long-chain hydrocarbon films involved in the boundary lubrication of metallic surfaces. I also derived a theory that explained the electron diffraction patterns obtained from the oriented monolayers.
In 1946 we both went to work permanently in Washington for the Naval Research Laboratory. Our interest continued in developing the quantitative aspects of gas electron diffraction analysis. The solution of a key problem that arose in such analyses had evident implications for crystal structure analysis and, in fact, other areas of structure determination. At about the time that these matters were developing, Herbert Hauptman joined us at the Naval Research Laboratory and we decided to pursue the implications for crystal structures. This eventually led to the development of the direct methods for crystal structure analysis with the major part of the mathematical foundations and procedural insights established in the early 1950's.
While all this was going on and with hardly missing a step from her research activities, Isabella mothered three children, Louise in 1946, Jean in 1950, and Madeleine in 1955. Louise is a theoretical chemist, Jean an organic chemist and Madeleine is a museum specialist trained in geology.
The initial applications of the procedure for structure determination for centrosymmetric crystals involving probability measures and formulas derived from the joint probability distribution were performed in the middle 1950's in collaboration with colleagues at the U.S. Geological Survey. Then, in the second half of the 1950's, through the efforts of Isabella Karle, an experimental X-ray diffraction facility was established in our own laboratory.
During the 1960's, there was an intensive program in my laboratory to develop a procedure for crystal structure determination of broad applicability that would encompass noncentrosymmetric as well as centrosymmetric crystals. Largely through the efforts of Isabella Karle, such a procedure was developed and called the symbolic addition procedure. This procedure had its origins in the theoretical work and the experience in practical application of the 1950's, but it also required some new procedural insights and some additional theoretical work to make it efficient and broadly applicable and avoid the pitfalls that easily arise when optimal pathways through a procedure must be chosen on the basis of probability measures. The first application of the symbolic addition procedure was published in 1963 and the first essentially equal atom noncentrosymmetric crystal structure to be solved by direct phase determination was published in 1964. This was followed by a number of exciting applications and toward the end of the 1960's many laboratories started to become interested in the potential of the direct method for structure determination.
During the 1960's, I collaborated with Isabella in some of her investigations and derived with her a variance formula that was the basis for applying probability measures to procedures for analyzing noncentrosymmetric crystals. In addition, I also carried out a number of theoretical investigations. Perhaps, the most useful one concerned a procedure for developing a fragment of a structure into a complete one by use of the so-called tangent formula for phase determination.
During the 1950's and 1960's, I maintained an interest in gas electron diffraction and made some experimental and theoretical studies of internal rotation and coherent diffraction associated with excitation processes. The latter was especially interesting, but required extensive experimental development that the resources available to me did not permit.
In the 1970's, I continued theoretical work in crystal structure analysis that included the derivation of a "tangent formula" for phase determination that was based on the more restrictive higher and higher order determinants from the determinantal inequalities. I showed how joint probability distributions relevant to crystallographic quantities could be put into an exponential form and thereby decrease considerably problems with asymptotic convergence. I also derived heuristic joint probability distributions based on the determinants involved in the determinantal inequalities and obtained from them formulas for evaluating triplet phase invariants and, later on, formulas for the expected values of phase invariants and embedded semi-invariants of any order, triplet, quartet, quintet, etc. The utility of phase invariants of high order in phase determination has so far been rather limited, except perhaps collectively in the high order determinants where they have been useful for refining the values of approximately determined phase values.
I participated with Wayne Hendrickson of my laboratory in some refinements of macromolecular structure with the use of the tangent formula and also had some early participation with John Konnert and Wayne Hendrickson in the constrained refinement technique for macromolecules. In collaboration with John Konnert and Peter D'Antonio, procedures were developed for determining atomic arrangements in amorphous materials based on criteria similar to those applied to molecular vapors. Collaborations on structural problems also included Judith Flippen-Anderson, Clifford George, Richard Gilardi and Alfred Lowrey.
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This is my BrainyGoose:
United States, IL, Chicago, English, Italian, Genry, Male, 21-25, bodybulding, swiming.