thingiverse
pmoews
Retired scientist protein crystallographer Reminisces of Enzymes and an Ah Ha Moment Cleveland - Western Reserve University In the mid 1960's the atomic structure of the enzyme lysozyme was determined by x-ray crystallography at The Royal Institution in London. A new technique, "the heavy atom method", was use to directly calculate a 3 dimensional electron density map of the enzyme. It was the first enzyme to have its structure determined. At that time I was an assistant professor of chemistry at Western Reserve University. Denied tenure I set out to become a crystallographer. I hoped to join one of the groups at The Royal Institution, learn protein crystallography, return to the United States, and start a protein crystallography group. I had some experience with x-ray crystallography as one of my doctoral committee members, J. L. Hoard, was a crystallographer and I had watched his students collect data and solve structures. I bought a set, 3 volumes, of the International Tables for X-Ray Crystallography - 1965 edition - and began to study crystallography. My first task was to determine the structure of a crystal and publish a paper about it. I looked through old German inorganic literature for a suitable compound. It needed to have a simple structure with few structural parameters, require least squares analysis for its solution, and therefore be illustrative of most aspects of modern crystallography. After a few trials I choose potassium hexachloromanganate, a cubic structure with a single structural parameter, as my crystal. The chemistry department was equipped with x-ray cameras and a mini 4 circle mount for single crystal work. I chose the GE powder diffractometer as it allowed more accurate error measurements. I struggled with the least squares analysis but after reading a number of early crystallogrqphic papers set up an algorithm using paper and pencil. I arranged columns of numbers in notebooks with multiplications across the pages and additions top to bottom; cycle after cycle of refinement. Then a lucky break, I took a course in Fortran programming using Western Reserves GE 225 computer. The algorithm I used to set up my notebook method was easily transferred to Fortran. I was able to quickly check my work and also able to add additional parameters; separate temperature factors for the potassium, chlorine, and manganese atoms. My paper, a single author one, was published in Inorganic Chemistry in 1966. "The Crystal Structure, Visible, and Ultraviolet Spectra of Potassium Hexachloromanganate(IV)", P. C. Moews, Jr, Inorganic Chemistry 1966, 5, 1, 5 - 8 London - The Royal Institution I then applied to the National Institute of Health for a fellowship to study enzyme crystallography at The Royal Institution. To my surpise my application was quickly accepted . The salary was at the level of a senior postdoctoral student and included overhead funds for The Royal Institution. I joined Charles Bunn's group at The Royal Institution in the fall of 1966. Bunn was trying to determine the structure of the enzyme rennin. Rennin is used commercially to make cheese and is extracted from the stomach of the calf. Bunn had crystals suitable for crystallographic study which he grew from aqueous solutions of rennin by slowly increasing the concentration of sodium chloride. My first task was to make the heavy atom derivatives which are necessary for determining the phases of the xray structure factors found from diffraction patterns of the native enzyme. The group that had determined the structure of lysozyme, led by David Phillips, was leaving the Royal Institution and a new director, George Porter, had taken the place of Sir Lawrence Bragg. David Phillips was made a professor of molecular biology in the department of zoology at Oxford. He left enough crystallography equipment so that Bunn and I could continue work but took the Elliot 803b computer to Oxford promising to return it within 2 years. While the computer was gone I was given access to a similar machine at Kings College, London and also the use of an Argus Ferranti machine at Perutz's laboratory in Cambridge. I was allowed to use the Argus Ferranti machine starting about 4 pm on Friday's till I had to leave to catch the last train back to London. Fortunately the Elliot 803b machine was returned in less than 2 years as promised. I believe I was well treated because I worked with Charles Bunn who was a friend of Perutz and also because I brought funds to the Royal Institution. As well I helped some of George Porter's people with programs for the Elliot. One member of David Phillips group, Tony North, spent the best part of a day with me going through the code for one of their principal programs, "Pangloss". It was so called because it did all things for the lysozyme problem. This was extremely useful for me and I was easily able to convert the program to work with the space group for the rennin problem. Bunn's laboratory was on the fourth floor of the Royal Institution. It included a chemical laboratory with a hood, equipment for making density measurements on x-ray films, and two offices. one his own and one for students. He added a desk in a corner of his office for me. The x-ray laboratory and computer were in the basement. It contained a dark room, three Super precession cameras, and a room sized high voltage power supply surrounded by a Faraday cage. Three additional precession cameras made by Stoe were added a year or so later. The x-ray laboratory has once been used by Michael Faraday. There was a covered hole in the floor where he kept frogs for his electrical experiments. Shelves along the wall, paralleling Albemarle Street, were used to hold the Daniels cells which were Faraday's source of electricity. The sink in the dark room was very old; carved from a single block of stone. Faraday's great magnet was stored in an adjacent room. The Royal Institution was constructed before electricity was wired into buildings and before electric motors were invented. The fourth floor and basement were connected by an elevator than was activated by water pressure. A full time electrician was employed to keep things running. He and his wife lived in a small house that was built on the roof of the Royal Institution. Bunn and I had lunch together almost every day. Bunn was a vegetarian having decided as a young man that it was morally wrong to eat animals. He however did eat cheese and milk. We dined at a pleasant nearby vegetarian restaurant. Perutz came for lunch at intervals of about a month. As Perutz was lactose intolerant we had to find another restaurant. Conversations were rather formal, Bunn and Perutz addressing each other by their surnames. Royal Institution tea ladies served tea and biscuits every afternoon. Most of the staff came for tea; it was served in a large comfortable room on the second floor. The tea was brewed in large tea pots and served with biscuits, usually plain ones. I was introduced to Sir Lawrence during one of the teas, He was treated with deference by all. The tea and biscuits were replaced by champagne and smoked salmon when George Porter received the Nobel Prize. For the first 2 or 3 years we tried to make heavy atom derivatives of rennin; over 100 attempts in all. No luck and my fellowship ran out. Bunn provided me with funds from the Medical Research Council of Great Britain so that I could continue work. We decided to expand out efforts to include other enzymes of a similar nature; enzymes that had been studied and used in the manufacture of cheese. Two such enzymes were provided to us by cheese makers; one from Endothia parasitica, a second from Mucor pucillis. We were able to purify and crystallize these enzymes and started looking for heavy atom derivatives. Again no joy. Bunn reached retirement age as I finished my sixth year at The Royal Institution. A brief discussion of the rennin work in included near the end of Bunn's obituary. https://royalsocietypublishing.org/doi/pdf/10.1098/rsbm.1991.0004 When Bunn retired I was again unemployed. I kept on working in lieu of something else to do. Eventually I was called in to George Porter's office and he told me that I really had to go; he added that he would give me a month's pay out of "his own funds". Of course I had been looking for somewhere to go and had written many many letters with no results. There were few university positions available in the United States; US universities were beseiged by antiwar demonstrations and scientific studies had started a slow decline in importance and support. As well the English postal system went on strike and it was difficult to communicate with the US. I was offered a research post at the University of Edinburgh but finally accepted a post doctoral position with Robert Kretsinger at the University of Virginia in the fall of 1972. Charlottesville - University of Virginia Kretsinger and Nockolds had just published the structure of a small protein, 107 amino acids, carp muscle calcium binding protein. Evidently some doubted the correctness of the work - University of Virginia - really! -, which upset Kretsinger. He wanted me to refine the structure using standard crystallographic methods to confirm its correctness. Also he was to spend a sabbatical year in Sweden and needed someone to watch over a student. The University of Virginia had recently installed a CDC6400 computer, a state of the art machine which I had access to. However no structure containing more than 100 amino acids had been refined by least squares methods at that time and the CDC6400 machine, while fast, was not fast enough to do least squares refinement. I choose to refine the structure by a combination of difference fourier analysis and model building using some of the techniques contained in the program "Pangloss". The work went well and one of Kretsinger's acquaintances, Wayne Hendrickson at the Naval Research Laboratory, became interested, and made helpful comments. He added an appendix to the paper. "Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis", P. Moews, R. Kretsinger, Journal of Molecular Biology, 1975, 91, 201-228. University of Connecticut After two years the money again ran out and I needed a new job, hopefully one that might be more permanent. I found a job as a research assistant at the Institute of Materials Science at the University of Connecticut. The Institute of Materials Science was contained in a building on the campus which held the Physics and Mathematics departments with offices and class rooms. The director was Lee Azaroff, one of Martin Buerger's students. Martin Buerger is the inventor of the x-ray precession camera. The institute contained a well equipped x-ray diffraction labratory. The building also held the main computer at the University of Connecticut, an IBM 360. My task was to help a young faculty member, Jim Knox, a member of the biology department. He had an office and a small laboratory on an upper floor of the institute, but most of the work was done in the x-ray diffraction laboratory on the ground floor. One room there held a large central table, surrounded by stools, one of which I used as my desk. I piled my paperwork, computer output, etc. on the table. Early on in my stay at UC Knox sent me to a conference in San Francisco There I heard a talk by a young physicist from Stanford, he wore a leather jacket as I recall - and talked about generating a tuneable monochromatic x-ray beam from the radiation emitted at a bending magnet on a particle accelerator. The strength of the beam was about equal to that of an x-ray tube. It did not seem that important at the time but this turned out to be a revolutionary advance. The beam intensity could be greatly increased and similar sites were built across the world. I made many trips to such sites to collect data. Knox had a student, a woman, working on a doctorate, Judy Kelly. She was the wife of a professor in the art department and had worked, I believe, before marrying and resuming study. We became coworkers and friends. She rose rapidly at the University eventually becoming chairman of the biology department. Knox sometimes seemed to find it difficult to give me orders and at times he told Kelly what he wanted me to do and she passed the orders on to me. Knox was working on the structure of an enzyme, the penicillinase excreted by E. coli bacteria. The enzyme destroys penicillin and protects the bacteria. Knox had determined a low resolution structure that lacked detail. Only the overall shape of the molecule was seen; not enough detail to trace the alpha carbon chain. It was proving difficult to grow crystals of higher quality so as to increase the resolution of the structure. This was an ambitious undertaking and there were competitors. A group at Oxford led by David Phillips was working on the penicillinase excreted by B. cereus and several groups were working on the penicillinase excreted by Staph aureus Knox sent me to give a paper at a conference on Holy Island off the east coast of Scotland. See Journal of Molecular Biology, 1976, 104, 865 - 875. This was an unusual place for a conference, the island is only accessible from the shore at low tide. I believe the organizer held the conference there to aid the hotels and restaurants as it is a tourist destination. I refused the honorarium, for some reason, I recall hearing that David Phillips had remarked he was going to "put fresh blood" on the Bacillus cereus problem. Shortly after that time a shock - the structure of the penicillinase from Staph aureus was determined and would soon be published; progress too on the penicillinase from bacillus cereus. At about this time these types of penicillinases were renamed beta lactamases. Getting the structure of the beta lactamase from E. coli was now less important, especially as it seemed that it might not be possible. For a few days turning to the cheese making enzymes that I had worked on was considered and then dropped. I began to think that perhaps I should quit research and get a real job. I interviewed Parker Brothers in Beverly, MA and gave a seminar about microprocessor based toys using a simple language suited for the purpose. This resulted in a call from Azaroff who increased my salary by $1000 a year. Then a near miracle. I was a bystander to this event and will describe it as I recall it but was not aware of all the details and my account may contain errors. Perhaps romanticized from my observations and the few facts available to me. An enzyme conference was held at Cambridge that we all attended. Professor Ghuysen, who directed an enzyme group at the Universite de Liege in Belgium, also attended. He described an enzyme, a transpeptidase, which was poisoned by trace quantities of penicillin. It was found in soil samples collected in the Belgium Congo and referred to as "R61", evidently from a label on the soil sample container. His group had rented a pilot plant in France, grew the micro organism in large tanks, and had gram quantities. Ghuysen evidently did not want to give samples of R61 to cystallographers but wished to collaborate with crystallographers and train his own people. I saw Ghuysen and Judy Kelly talking while taking a boat ride in a Boston park. A day or two later Ghuysen came to the University of Connecticut and I again observed them in a long conversation taken in a cemetary across the street from the Institute of Materials Sciece. A collaboration was started; Judy was given samples of the purified enzyme. During the next few summers Belgians arrived at the Institute. All spoke better English than most Americans and of course all also spoke French. There was even one who spoke Walloon. They seemed to have a good time, the first ones bought a car, which was passed on from person to person. Early in the summer of 1982 Otto Dideberg and I think Jean Marie Frere arrived. Otto had a tape written for our IBM computer which contained phased structure factors for the R61 enzyme. Unfortunately he had made a mistake when writing the tape. IBM tapes at that time had a number of formats, two were VS and VSB, variable span, and variable span blocked. Otto had written the tape in either VS or VSB format but then used the other format when describing the tape. It was unreadable. My son David wrote an assembly language program which read the problem tape and output a valid one. I believe I calculated the map using a calcomp plotter to output contours to be plotted on sheets of transparent plastic and stacked so that details about the molecule could be observed. The Ah Ha moment occurred the morning after the sheets had been traced and stacked. We all gathered around the stacked sheets to view the map. Jim Knox exclaimed: "Look there are 3 alpha helices guarding a beta pleated sheet; it looks a lot like a beta lactamase!" As R61 is poisoned by penicillin rather than destroying it, it must be the target of penicillin; that is the enzyme that helps construct cell walls. A paper was soon written and submitted to Science. Titled "Penicillin Target Enzyme and the Antibiotic Binding Site" it appeared in the October 29th 1982 issue. A reporter from the Hartford Courant, Joel Lang, interviewed us and his article appeared in the February 6th 1983 edition of the Northeast Magazine, a Sunday Supplement of the Hartford Courant. The title of the article was featured on the cover - "Unraveling the Great Penicillin Mystery". Ah Ha moments are rare; life went on. Protein crystallography was revolutionized during the eighties. Sealed tubes and rotating anodes took back seats to synchrotron sources. Film was replaced by area detectors. Stacks of transparent plastic containing contours were replaced by three dimensional display terminals. Computers were continuously upgraded, the IBM 360 at the University of Connecticut was replaced by a 370/168, then a 3081D, 3084, 3090, etc. Somewhere late in the eighies I was given a set of structure factors obtained from a crystal of a beta lactamase generated by Bacillus licheniformis. The data was collected by the Belgians but I believe they were having difficulties and Knox handed the native data set over to me. One difficulty was that there were two molecules in the asymmetric unit, that is the minimum repeating unit. I was able to solve the structure using model building with rotation and translation functions. My son David helped me understand the angles of rotation used to refine and describe the structure. Some of his suggestions are included as an appendix to a paper describing the solution. "Beta-Lactamase of Bacillus licheniformis 749/C at 2 Angstrom Resolution", Paul C. Moews, James R. Knox, Otto Didberg, Paulette Charlier, and Jean-Marie Frere, PROTEINS: Structure, Function, and Genetics 7: 156-171 (1990) Lack of funds caused me to retire from the University of Connecticut on October 1, 1996 at the age of 63. I was rehired on a part time basis and received a small salary together with my pension until the summer of 1999. I continued to occupy my place at the x-ray laboratory for a few additional months without being paid. Paul Moews - 2024
- Relation
- Ingen
- Commercial signal
- None
- Social links
- Ingen fundet
- Analyse
- Ingen native Thingiverse commercial license signal fundet på profile page.
- Sidst set
- 2026-07-11 08:20
- Analyseret
- 2026-07-12 20:37
Stats
Platformprofiler
| Platform | Profil | Stats | Analyse | |
|---|---|---|---|---|
| thingiverse | pmoews 14952 |
18,245 følgere
585 modeller
- downloads
9 likes
|
Ingen native Thingiverse commercial license signal fundet på profile page. 2026-07-12 20:37 |
Licensplatforme
Ingen commercial licensplatforme fundet endnu.
Indekserede modeller
1 modeller| Model | Kategori | Licens | Kandidat | Sidst set |
|---|---|---|---|---|
| Seated Figure - Wood Carving 7378837 | Scans & Replicas | Creative Commons - Attribution | dormant Opportunity 29 | 2026-07-11 08:20 |