From reindeers to synchrotrons: Personal recollections

G. Semenza and A.J. Turner (Eds.) Selected Topics in the History of Biochemistry: Personal Recollections VIII (Comprehensive Biochemistry Vol. 43) 9 2004 Elsevier B.V.

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Chapter 3

From Reindeers to Synchrotrons: Personal Recollections CARL-IVAR BRAND]~N

Karolinska Institute, Microbiology and Tumour Biology Centre, Box 280, SE-17177Stockholm, Sweden

Abstract My first years in science were spent as an inorganic chemist at Uppsala University studying adducts between Lewis acids and bases by X-ray crystallography. A postdoctoral year in Cambridge at LMB converted me from a rather ignorant chemist to a devoted molecular biologist and I have studied structure and function of proteins ever since. The first major project after returning to Uppsala focussed on alcohol dehydrogenase for which we could postulate a detailed mechanism of action based on structure determinations of a number of complexes in two different conformations. The second major project involved Rubisco, the enzyme that catalyses the initial carbon dioxide fixation in green plants. Towards the end of my scientific life I spent five years as research director of the European Synchrotron Research Facilities in Grenoble, France. I was responsible for building up experimental facilities for chemistry, biology and medicine and managed to promote strong facilities for structural biology. In my spare time between these activities I wrote a textbook on protein structures with

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J o h n Tooze, for m a n y years coeditor of the journal S t r u c t u r e with Wayne Hendrickson a n d later A l a n Fersht a n d I have spent a large fraction of my time on m a t t e r s of science policy a n d advice.

The First Years I was b o r n on 14 May 1934 in a tiny village in L a p p l a n d at the far n o r t h of Sweden. My father, H.M. Br~ind~n, was t h e local school teacher a n d I spent my first six years at school u n d e r his supervision. We were only about 15 children in different classes in the same classroom, so while my father was t e a c h i n g one age group the other pupils were studying on their own. In retrospect I have realised that this was a n excellent arrangem e n t since I learnt at a n early age to concentrate on t h e work at h a n d disregarding the noise a r o u n d me. The village was poor, scholarly p u r s u i t was u n h e a r d of, a n d the climate consisted of nine m o n t h s of winter a n d t h r e e m o n t h s of cold wind. But n a t u r e was wonderful w i t h beautiful lakes w i t h plenty of fish a n d deep forests full of berries a n d m u s h r o o m s a n d trees to climb. Our village was along the trails t h a t the reindeers took in late a u t u m n when they were herded from the m o u n t a i n s to the coast. It was a wonderful experience to be in the middle of a herd of 10,000 reindeers m i n d i n g their own business a n d ignoring a young boy. The appreciation of this type of n a t u r e a n d such experiences have r e m a i n e d w i t h me for my whole life. However, already at the age of twelve I realised t h a t I did not w a n t to spend the rest of my life in this place a n d that my only chance of getting away was to obtain an education. E n c o u r a g e d by my father, who paid the costs out of his small salary, I left home at the age of t h i r t e e n to a t t e n d s e c o n d a r y school about 50 miles from the village. In those days 50 miles was a long distance, so I h a d to rent board a n d lodging w i t h a family together with two other boys at the school. In order to save money my father h a d supervised me in studying the

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Fig. 1. The author at the age of 69.

first-year course at home. I was very nervous d u r i n g the two-day examination, b u t w h e n I was told t h a t I h a d passed a n d could s t a r t school the next week I experienced one of the happiest days of my life. My d r e a m s would be realised, I could study instead of doing m a n u a l labour a n d I might even be able to see more of the world t h a n Lappland. W h e n I was sixteen my mother, G.M.E. Br~ind~n, h a d enough of the darkness a n d isolation of Lappland, so the family moved to the south of Sweden, to her home village on the west coast. I c o n t i n u e d my studies for t h r e e years in Uddevalla, a small shipping a n d military t o w n t h a t had been strongly c o n t a m i n a t e d w i t h fascist activities d u r i n g the second world war. Needless to say, intellectual p u r s u i t was not in very high e s t e e m a m o n g the citizens b u t we h a d some very good teachers at the school, especially in mathematics which was my favourite subject. Again, the school was about 50 miles from where my p a r e n t s lived, so

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I h a d to r e n t board and lodging in Uddevalla. The move from Lappland h a d d r a i n e d my father of money, so I h a d to t a k e a b a n k loan to pay for the rent. School was of course free as all education in Sweden still is. My mathematics teacher advised me to continue my studies at university, to take a degree in m a t h e m a t i c s a n d physics a n d become a teacher at high school like himself. In those days one could choose between biology a n d a special course in higher mathematics even t h o u g h the school was supposed to give everyone a general education in all branches of science. I chose the m a t h e m a t i c s course w i t h the consequence that I was never t a u g h t any biology at school which is somewhat ironic since I have published about a h u n d r e d scientific papers in biological journals. This illustrates that formal t r a i n i n g in specific subjects at school has little relevance to what you do later in life. After finishing school a n d 15 useless m o n t h s of military service, I could see no financial possibility to continue studies at the university a n d was considering various other alternatives. At that crucial point in my life, the socialist government a n n o u n c e d that the best students with no m e a n s of support could obtain a fellowship covering free board and lodging for studying at the university. I applied, obtained the fellowship a n d went to study at Uppsala University.

An Inorganic Chemist in Uppsala Influenced by my teacher I decided to study for a major in mathematics a n d a m i n o r in physics when I came to Uppsala. The mathematics studies went quite well but I had problems with physics. In order to be eligible to start studies on physics I h a d to take a propedeutic course in classical mechanics which I found so b o r i n g t h a t I could not force myself to continue after the first couple of lectures. In desperation I began to study chemistry instead, although I h a d never liked chemistry at high school. To my surprise I found t h a t I enjoyed chemistry at

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the university level a n d reading the textbooks by Linus Pauling, "General chemistry" and "The n a t u r e of the chemical bond': was a n intellectual feast. After a couple of weeks in the laboratory I was approached by the acting c h a i r m a n of the d e p a r t m e n t of inorganic chemistry, Ingvar Lindqvist, who asked me if I was interested in doing a PhD in his group w h e n I h a d completed my chemistry course. It t u r n e d out that it was very rare t h a t a major in mathematics s t a r t e d chemistry courses, a n d since the major t h r u s t of his research was X-ray crystallography, he was eager to obtain students with a mathematical background. I was of course extremely flattered by his offer and accepted w i t h o u t hesitation. I h a d never d r e a m e d of being offered a possibility to pursue a research career, but realised t h a t this was both an o p p o r t u n i t y a n d a challenge. W h a t I did not realise t h e n was that, since I took that decision to go into science, I have never h a d a single b o r i n g day. Later in life I repaid Lindqvist's offer by accepting as PhD students two physics undergraduates, J o h a n Aqvist and P~ir Nordlund, who were among the first batch of physics students in Uppsala t h a t combined physics with biotechnology. J o h a n is now a professor at Uppsala University a n d P~ir a professor at Stockholm University. The research projects in this group were centred a r o u n d t h e properties of proton-free solvents such as phosphorous oxychloride, POC13. The c o m m o n belief was t h a t a metal halide t h a t was dissolved in such a solvent formed ionic species. My first project was to prepare, crystallize a n d determine the X-ray s t r u c t u r e of the c o m p o u n d formed between SbC15 a n d POCl~ to verify t h a t it was built up from SbC16- and POC1 + ions. To the surprise of everyone in the field I could show t h a t it was not a n ionic comp o u n d b u t a coordination adduct with the oxygen atom of POCI3 b o u n d to Sb [1].This discovery c h a n g e d the focus of the research group a n d I subsequently solved the s t r u c t u r e s of a n u m b e r of adducts between Lewis acids a n d Lewis bases. I was very lucky to have a full-time research position which was paid for by a grant given to Lindqvist from the US Air Force Office of Scientific Research a n d Development Command. They

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distributed money for basic research after the l a u n c h of the Soviet satellite S p u t n i k in order to increase the science base in the w e s t e r n world. D u r i n g this period I obtained a fair knowledge of crystallographic methodology which has subsequently been very useful. C o m p u t i n g in those days was very primitive a n d t i m e consuming. S t r u c t u r e factors were calculated by using m e c h a n i c a l calculators to add p r e c o m p u t e d sine a n d cosine functions listed on so-called Beever-Lipson strips. We h a d a home-made analogue c o m p u t e r for Fourier summations, the H~igg-Laurent machine, where the three-dimensional Fourier sums were b r o k e n down into t h r e e successive one-dimensional s u m m a t i o n s (Figure 2). In 1960 I h a d about h a l f a dozen solved structures, but they were n o t refined due to the t i m e - c o n s u m i n g calculations. However, d u r i n g the s u m m e r I a t t e n d e d a s u m m e r school in M a n c h e s t e r on m o d e r n m e t h o d s of X-ray crystallography organized by D.W.J. Cruickshank. In a series of lectures he described how the m e t h o d of least squares could be applied to the refinement of X-ray s t r u c t u r e s a n d also outlines of his p r o g r a m for one of the first digital c o m p u t e r s in UK. One of t h e p a r t i c i p a n t s was Stig A s b r i n k from Stockholm University, a n d one late evening we decided over a bottle of whisky to join forces a n d write a similar p r o g r a m for the first Swedish electronic computer, BESK. This c o m p u t e r h a d a m e m o r y of 1000 words, was equipped w i t h a larger magnetic d r u m m e m o r y a n d used p a p e r t a p e for i n p u t a n d output. There was no compiler for a higher language, so the whole p r o g r a m was w r i t t e n in machine code. It took me six m o n t h s to write a detailed flow chart, four m o n t h s for A s b r i n k to write the m a c h i n e code a n d one year for the two of us to debug the program. Over t h e next t e n years this p r o g r a m m e was u s e d by t h e whole S c a n d i n a v i a n crystallographic c o m m u n i t y a n d was n u m b e r one in the use of c o m p u t e r t i m e for b o t h BESK a n d its m u c h improved successor FACIT. In a few m o n t h s I h a d refined my s t r u c t u r e s a n d s t a r t e d to prepare for my PhD thesis. By t h a t t i m e I h a d b e g u n to

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Fig. 2. The author and a cotleaguz, Arne Hansson, in flont of an analogue computer, specially designed to calculate Fourier summations. This machine was used at the chemistry department of Uppsala University during the 1950s before digital computers became available. It was designed by the head of the department, Gunnar Hcigg, and a professor in engineering sciences, Torbern Laurent, who was the father of one of the authors in Vol. 42 of Comprehensive Biochemistry, Torvard Laurent.

realise t h a t the work I h a d done after my first s t r u c t u r e was more s t a m p collecting t h a n real science. W h e n I h a d chosen the subject for my PhD thesis, I h a d not yet come across Francis Cricks advice to young scientists: "If you ask i m p o r t a n t questions, you obtain i m p o r t a n t answers': I had decided to leave science after my PhD a n d start w h a t is nowadays called a software c o m p a n y w r i t i n g commercial programs for BESK. Such programs were in great d e m a n d a n d there were very few people a r o u n d w i t h the relevant experience. However, before I could get my PhD I had to t a k e one more course in chemistry. I opted for biochemistry since I knew absolutely n o t h i n g

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about the subject. This decision completely changed my plans for the future. I realised that I could apply my knowledge in crystallography to scientifically important and intellectually stimulating problems in biology. I wanted immediately to move into the field of protein crystallography, and the obvious place to learn the trade was the MRC Laboratory for Molecular Biology, LMB, in Cambridge, UK. My supervisor, Ingvar Lindqvist, encouraged me and was able to rapidly arrange a place for me in Cambridge. By chance he met John Kendrew at a meeting in the Vatican who told him that he was looking for someone with experience of computing in crystallography who would be willing to join his group. Bror Strandberg, who subsequently started his own structural biology group at the Biochemistry Department of Uppsala University, had recently left LMB after taking part in the high-resolution structure determination of myoglobin.

In Cambridge 1962-1963 I arrived in Cambridge in May 1962 and my wife Lisbet (L.M. Br~ind~n) joined me shortly afterwards. LMB was in those days one of the most exciting and stimulating places for a young postdoc to spend some time. Max Perutz, who had built-up the laboratory with an incredible ability to choose the right people and let them work in independence, was working on the highresolution structure of haemoglobin. Once in a while Jaques Monod visited to lecture and discuss with Perutz his latest development on the theory of allosteric transitions for which the conformational changes in haemoglobin played an important role. Francis Crick and Sydney Brenner were working on the genetic code and attracted an almost endless stream of visitors who gave seminars on their latest work. Fred Sanger's group was sequencing several large proteins and interacted frequently with the structural biologists. Aaron Klug had just moved in from London and was developing his methods of

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optical filtering a n d three-dimensional r e c o n s t r u c t i o n of elect r o n micrographs. The day-to-day interactions with the postdocs in s t r u c t u r a l biology provided me with a n e t w o r k of friends from which I have benefited ever since. Michael R o s s m a n n a n d David Blow were developing the m e t h o d of molecular replacement, Lubert Streyer was t r y i n g to demonstrate t h a t one could see the difference b e t w e e n water a n d cyanide b o u n d to iron in a difference Fourier synthesis of myoglobin, Allen E d m u n d s o n was d e t e r m i n i n g the amino acid sequence of myoglobin a n d Richard Henderson h a d joined David Blow to determine the s t r u c t u r e of chymotrypsin. I rapidly engaged myself in w r i t i n g a c o m p u t e r p r o g r a m for the refinement of myoglobin by the m e t h o d of least squares. The s t r u c t u r e h a d been solved a few years earlier and now more accurate a n d higher-resolution data had been collected by David Phillips at the Royal I n s t i t u t i o n in L o n d o n using his newly developed linear diffractometer. J o h n Kendrew a n d H e r m a n Watson were using these data in combination w i t h approximate coordinates of the myoglobin s t r u c t u r e for refinement by Fourier synthesis but better methods were needed. Since myoglobin was the only p r o t e i n s t r u c t u r e t h a t h a d been solved, there was virtually no experience of refining protein structures. A previous attempt, using only diagonal terms and no constraints, to ensure k n o w n geometry of rigid groups h a d failed. In collaboration w i t h Ken Holmes I designed a p r o g r a m using exact constraints a n d a 6 x 6 block diagonal matrix. It t u r n e d out to be a non-trivial problem to adapt this m e t h o d to available computers. Even t h o u g h we were using the fastest available comp u t e r s in UK, IBM 709 a n d later 7090, we had to invent several tricks to reduce c o m p u t i n g time and storage space. The final version of the p r o g r a m required 12 magnetic tape stations for intermediate storage. W h e n the p r o g r a m was finally finished we e s t i m a t e d t h a t each cycle of refinement would require 16h of c o m p u t i n g time on the 7090 at the IBM c o m p u t i n g centre at a considerable cost. We were allowed to r u n only one cycle a n d t h e n evaluate the results. W h e n I h a d booked the time and

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requested the 12 magnetic tape stations (which was the most expensive booking the IBM computing centre had ever experienced), I happened to overhear a conversation in the users' office at the centre about what "the crazy Swede" was up to. The r u n was successful computationally but the result was disappointing. The structure had improved only marginally and we were very disappointed. In retrospect we know that this always happens during the first few cycles of a least-squares refinement; the structure is shaken up before it rapidly falls into a local minimum. However, it was a good try in those days but it was ten years before its time. For certain political reasons at LMB this work was never published except as an abstract to a lecture I gave at the IUCr meeting in Rome in 1963 [2]. During my stay at LMB Crick, Watson and Wilkins obtained the Nobel Prize in medicine and Perutz and Kendrew obtained the prize in chemistry. The party given by Crick in his home, The Golden Helix, is memorable both for the fireworks arranged by one of the students in the lab, Mark Bretcher, and for the amounts of champagne served. One of the rockets in the firework display misfired and ended up in the dog kennel of Cricks neighbour frightening the dog out of his wits. During the party, celebrating the chemistry prize, Perutz made a speech where he said that the reason why he and Kendrew had succeeded where others had failed was that they had been so ignorant about crystallography that they did not know it was impossible to solve a protein structure. Knowledge is fine, but too much is inhibitory for breakthrough science. The year I spent at the LMB has had a profound influence on my scientific life. I became converted from a scientifically rather ignorant inorganic chemist to a devoted molecular biologist. I became acutely aware of the difference between pedestrian science and working on the frontiers of scientific discovery which has strongly influenced not only my own science but also my engagements in different science policy matters and scientific advisory boards. I realised the importance and the joy of working out concepts and details of molecular aspects of

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the biochemistry of living organisms, and finally, I became integrated into a network of structural biologists who have at one time or another been at LMB and many of whom have become close friends.

Building up a Group in Structural Biology in Uppsala I came home from Cambridge in May 1963 to a 6-year research position that Ingvar Lindqvist had a r r a n g e d for me. He had moved from Uppsala University to a chair in chemistry at the Agricultural University in Uppsala and wanted to provide the opportunity for me to build up a group in structural molecular biology within the chemistry department. The Agricultural University reluctantly agreed to give me a position but no additional resources, since molecular biology was not a recognised subject and furthermore was considered to be of no relevance for agricultural science. However, there were no other research positions available for me in Sweden and a grant from NIH provided the financial basis for starting the work. During these years I was teaching mathematics (which I enjoyed) to agricultural undergraduate students to motivate my position. Ingvar Lindqvist always supported me strongly and he subsequently became my neighbour and close friend. His bright daughter, Ylva Lindqvist, later became my PhD student and is now a professor at the Karolinska Institutet in Stockholm. During the last months at LMB, Michael Rossmann, Herman Watson and I had decided that each of us should determine the structure of a dehydrogenase enzyme to see if they formed a structural family similar to the globins. Rossmann, who was leaving LMB for Purdue University, started work on lactate dehydrogenase (LDH), Watson had already started on glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was being sequenced by Ieuan Harris in Sanger's lab, and I formed a collaboration with Hugo Theorell in Stockholm who was an expert

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on alcohol dehydrogenase (ADH). The collaboration w i t h Theorell lasted for m a n y years a n d I always enjoyed o u r interactions. In spite of his physical h a n d i c a p he was always cheerful a n d encouraging. His closest coworker in the lab, Ake Akeson, provided us w i t h m a n y grams of purified enzyme from horse liver over the long years we were working together. Building up a s t r u c t u r a l biology laboratory from scratch in those days a n d in t h a t place was not easy. Money was scarce; the N I H g r a n t allowed the p u r c h a s e of a microscope, a precession camera a n d an ordinary small X-ray generator with a sealed-off X-ray tube. There was no equipment or i n f r a s t r u c t u r e for biochemical work, not even a coldroom. The first crystals of ADH were grown in the k i t c h e n refrigerator of Ella Zeppezauer, my first r e c r u i t m e n t to the group. These a n d all our subsequent ADH crystals were very t e m p e r a t u r e sensitive a n d dissolved as soon as they were t r a n s f e r r e d to r o o m temperature. We therefore moved all the equipments into a small u n u s e d cold room located in a nearby building so t h a t we could grow the crystals, m o u n t t h e m a n d do the X-ray work in the same cold place. This worked so well t h a t all our subsequent studies were done in the same way until cryo-freezing with liquid nitrogen became standard. Having been brought up in L a p p l a n d I did not m i n d the long hours spent in the coldroom, but some of my coworkers from w a r m e r countries complained bitterly. I n those days there were n o t m a n y p r o t e i n crystallographers in the world a n d most of t h e m h a d been t h r o u g h L M B as postdocs. The first i n t e r n a t i o n a l s y m p o s i u m on m e t h o d s of protein crystal s t r u c t u r e d e t e r m i n a t i o n was held in 1966 in the ski resort Hirschegg in the Austrian alps. The m e e t i n g was organized by Max Perutz a n d Walter Hoppe from M u n i c h and had attracted 16 p a r t i c i p a n t s who comprised almost the entire comm u n i t y at t h a t time. It was an ideal size for intensive discussions on developing novel methods, b o t h experimental a n d theoretical. The afternoons were free for skiing a n d private discussions on the ski slopes with the consequence t h a t three of the participants, Michael Rossmann, R i c h a r d Dickerson a n d the

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secretary of Hoppe, were carried in on stretchers w i t h broken legs to the last session. The m e e t i n g became a biannual event for about a decade a n d has played an i m p o r t a n t role in the early development of protein crystallography. It took m u c h longer time t h a n I h a d expected to obtain a functional laboratory a n d an efficient group, b u t by the end of the 1960s it was all in place. In those days data were usually collected on film using precession cameras and the films were m e a s u r e d on a Joyce-Loebl microdensitometer. This i n s t r u m e n t recorded p e a k heights which were m e a s u r e d by a ruler. The results were not very accurate especially for w e a k reflexions, a n d novel methods p r o d u c i n g more accurate data were on their way. However, we m a n a g e d to produce heavy-atom derivatives, locate the heavy a t o m positions a n d by 1969 we h a d obtained a low-resolution s t r u c t u r e of ADH. Only a few weeks later I b r o u g h t a balsa-wood model of the electron density to a m e e t i n g in Konstanz, Germany, where I met Michael R o s s m a n n who h a d brought a similar low-resolution model of LDH. We c o m p a r e d the models a n d to our delight we could identify similar features in parts of the subunits. We were not believed by anyone even t h o u g h later high-resolution studies did show t h a t we had identified the correct regions of similarity. However, over a few beers we p e r s u a d e d the c h a i r m a n of our session, Dan Koshland, to give us time to present our c o m p a r i s o n so t h a t it would be recorded in the symposium book [3]. We immediately set out to obtain a high-resolution structure. By t h a t time an automatic computer-controlled film s c a n n e r was available at Stockholm University a n d we established collaboration w i t h Per-Erik Werner to evaluate our films using t h a t scanner. For almost two years we collected films, took t h e m to Stockholm to be s c a n n e d and brought back boxes of p u n c h e d cards which were processed at Uppsala University c o m p u t i n g centre. W h e n we finally calculated the high-resolution electron density map it was completely uninterpretable. By a more careful analysis of the data we found t h a t about 5% of the reflexions on each film h a d been m e a s u r e d incorrectly due to an error in

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the coordination of the filmscanner a n d its computer. Since this occurred in a r a n d o m way for native data, as well as t h e t h r e e heavy-atom derivatives, the net result was that 20% of the reflexions had a large error in the phase angles. I t h e n h a d to m a k e a very difficult scientific decision. Should we find a n o t h e r scanner a n d r e m e a s u r e the films or should we recollect all the data on our newly installed computer-controlled four-circle diffractometer? I chose the second alternative since I believed t h a t we would t h e n obtain more accurate data. W i t h i n a year we h a d recollected the data by r u n n i n g the diffractometer 24 h a day a n d 7 days a week. Since a crystal lasted only 6 - 8 h in the b e a m this involved frequent mounting, t e s t i n g a n d adjusting a new crystal i n d e p e n d e n t of the hour. This would have been impossible without devoted efforts of my group in which Hans E k l u n d played a major role. He later became an indep e n d e n t investigator in my department, has done excellent work on the enzyme ribonucleotide reductase a n d succeeded me as a professor a n d head of the d e p a r t m e n t w h e n I left Uppsala for Grenoble. We had now worked almost t e n years without significant results a n d without any s u p p o r t from the university except my salary for six years. Nevertheless, we had built-up a reasonably sized group a n d were fairly well equipped. How was t h a t possible? There were two agencies which consistently s u p p o r t e d me the whole time. One was the Swedish National Research Council, NFR, which fortunately h a d assigned Peter Reichard as the strong m a n for biochemistry in the chemistry board. He was a s t a u n c h s u p p o r t e r of my work and made sure t h a t I obtained adequate grants w i t h i n the constraints of the N F R budget, including my own salary when my position at the University was terminated. He t a u g h t me the i m p o r t a n c e of long-term s u p p o r t to young scientists who venture into new areas a n d who you believe will eventually m a k e progress. The second agency was a private foundation, the Wallenberg Foundation, which several times gave me money to buy expensive equipment, b o t h X-ray machines a n d later on expensive

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computers. Without these two agencies my group in s t r u c t u r a l biology would never have got off the g r o u n d in Sweden, a n d I would probably have accepted the offers to move abroad either to the Biozentrum in Basel or to Berkeley.

Alcohol Dehydrogenase I n t e r p r e t i n g the electron density from the new data gave me one of those rare m o m e n t s in the life of a scientist t h a t compensates for years of anxiety and h a r d work. The m a p was barely interpretable a n d I c o n c e n t r a t e d so h a r d to p u t the details into my b r a i n t h a t I h a d dreams d u r i n g the nights t h a t I was w a l k i n g a r o u n d in the map a m o n g the contours of the density. Finally, in the b e g i n n i n g of M a r c h 1973 I h a d traced a reasonable p a t h for the polypeptide chain in the d o m a i n t h a t b o u n d a coenzyme analogue and could write a topology diagram of the fold. I became ecstatic w h e n I c o m p a r e d the d i a g r a m w i t h the fold of the coenzyme-binding d o m a i n of LDH a n d found t h a t they were very similar. I h a d found the relationship between two members of the dehydrogenase family a n d discovered, s o m e t h i n g fundamentally new, t h a t the domains t h a t b o u n d the c o m m o n coenzyme, NAD, h a d similar folds despite no similarities in the amino acid sequences whereas the domains t h a t b o u n d the different substrates had different folds. I wrote an excited letter to Michael R o s s m a n n describing these results a n d predicted t h a t he would find the same p a t t e r n in GAPDH, the s t r u c t u r e of which he was in the final stages to determine. One m o n t h later he could confirm my prediction a n d coined the phrase "molecular fossils" for domains t h a t have r e t a i n e d the same function a n d similar folds, b u t whose amino acid sequences have diverged so m u c h t h a t no significant sequence identity remains. A few m o n t h s later we p r e s e n t e d these results at the I n t e r n a t i o n a l Congress of Biochemistry in Stockholm where I gave a plenary lecture in the m a i n hall in front of several thousand participants. S t a n d i n g there in front of a battery of at least

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20 microphones and with the front row 50 m away I realised t h a t I h a d come a long way from my childhood in Lappland. Shortly afterwards we jointly published a detailed description of these similarities and their implications for a possible early evolutionary relationship between these members of the dehydrogenase family [4]. A n u n e x p e c t e d consequence of these presentations were the large n u m b e r of invitations I obtained to lecture at seminars a n d meetings in Europe a n d US. These trips allowed me to be in touch with the developments in the field a n d to make m a n y friends for the rest of my life. D u r i n g this period our two sons were born, H e n r i k (C.H.I. Brfind~n) in 1965 a n d Per (RY.S. Brfind~n) in 1969. We were living in a big house on the college g r o u n d with a large garden close to a forest. This was a great place for children, lots of space both indoors a n d outside a n d flats nearby for s t u d e n t families with children. Both, my wife a n d myself, were working long hours a n d I guess the children grew a bit wild. D u r i n g the congress in 1973, Max Perutz, Dorothy Hodgkin a n d David Phillips came one day to visit my lab and to see the ADH structure. I h a d invited t h e m for lunch in our house, a n d since I was very anxious to give a good impression with these famous visitors, I h a d told the children t h a t for once they were not allowed to join us for the meal but should play in the garden. The weather was unusually hot t h a t day, a n d the visitors were e x h a u s t e d a n d i r r i t a t e d from the journey from Stockholm and a sightseeing t o u r of Uppsala in a hot car. They probably sensed t h a t I was tense, so lunch did not start in the best of moods. It did not become better w h e n suddenly pinecones came flying onto the table t h r o u g h the open window t h r o w n by my children in the garden. Later in life I could have used this incidence to relieve the tension a n d we could have had a jolly lunch, but I was in those days too insecure to try. I n t h e best British way o u r visitors p r e t e n d e d not to notice the strange disturbance a n d lunch c o n t i n u e d in a very s t r a i n e d atmosphere. Over the years I have since developed a strong friendship with all t h r e e of t h e m but we never m e n t i o n e d this lunch. In spite of the lack of sufficient

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time a n d attention I feel I have given my sons w h e n they were growing up, due to my devotion to science, we have developed a w a r m friendship over the years. Per is a high-school teacher in mathematics a n d philosophy, whilst H e n r i k took a degree in i m m u n o l o g y a n d has w r i t t e n textbooks in molecular biology a n d immunology; we have even w r i t t e n a p o p u l a r book together in Swedish on "Molecules of Life". Both have sons of their o w n now a n d the g r a n d c h i l d r e n are a c o n s t a n t source of joy for me. My engagement in ADH c o n t i n u e d for the next t e n years. We h a d showed at an early stage t h a t complexes c o n t a i n i n g coenzyme with or without substrate analogues p r o d u c e d crystal forms t h a t were different from that of the crystals we h a d used to solve the apoenzyme structure, presumably due to coenzymei n d u c e d conformational changes. Nowadays it would be simple to obtain this holoenzyme structure, collecting the data on a synchrotron in a few hours, feeding the data a n d the apoenzyme s t r u c t u r e into an automatic p r o g r a m suite, and seeing the elect r o n density of t h e holoenzyme appear on your c o m p u t e r display sitting on your desk in a few days. It was different 30 years ago. A complete description of t h e holoenzyme s t r u c t u r e to high resolution did not appear until 1981 [5]. Meanwhile, we studied a n u m b e r of inhibitors b o u n d to the apoform including b o t h substrate- a n d coenzyme-competitive inhibitors. F r o m these studies we deduced a m e c h a n i s m for the catalytic action of ADH [6], which in all essential aspects is still valid. W h e n we finally obtained the holoenzyme s t r u c t u r e we could show t h a t the conformational change consisted of a large rotation of the domains relative to each other which closed off the active site a n d shielded it from solution d u r i n g the catalytic action. In collaboration with M a r t i n Karplus we studied the dynamics of this d o m a i n rotation using c o m p u t e r simulations and found no significant energy barrier in r o t a t i n g the domains from one form to the other. Meanwhile, one of the Hugo Theorelrs students, Hans JSrnvall, d e t e r m i n e d the amino acid sequences of several different alcohol dehydrogenases a n d I enjoyed a very fruitful collaboration w i t h h i m building models of these

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enzymes. Homologous model building was in those days regarded with strong suspicion by the crystallographic community, but later experimental structure determinations have shown that our models as well as the functional implications derived from them were essentially correct. During this period we had frequent long-term visitors from outside Sweden who were engaged in chemical or biochemical work on ADH in their home laboratories. The most long-lasting and personally most rewarding such visitor was the biochemist Bryce Plapp, from Iowa, US. From careful kinetic studies of the catalytic reaction of ADH he designed conditions that enabled him to crystallise enzyme complexes containing both coenzyme and true substrates. The structure of one such complex showed unequivocally that the substrate was directly bound to the catalytically active zinc atom, an aspect of our suggested mechanism that had been the subject of considerable controversy between me and some members of the EXAF's community who claimed that they had evidence for watermediated binding. During his first stay with us in wintertime, Bryce learned cross-country skiing and became so good that after two months I had to stop smoking to be able to continue beating him. Unfortunately he went home, I took up smoking again and subsequently I have developed tumours in my lungs which will end my life somewhat prematurely. Jean-Pierre Samama was a young student who did his PhD work in our laboratory. He came from a group in the bioorganic chemistry laboratory of Jean-Francois Biellmann in Strasbourg, France, that worked on analogues to NAD substituted in the nicotinamide ring. During his stay in our lab he became so used to speaking and writing in English that when he presented his thesis at the University of Strasbourg, which he was forced to write in French, the department c h a i r m a n refused to approve it on the grounds that it was written in such a bad French language that he suspected that Samama neither had done any useful science nor had written any scientific paper. After a long discussion Biellmann and I together managed to

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convince h i m t h a t his suspicions were unfounded. S a m a m a is now a professor in s t r u c t u r a l biology in Toulouse a n d a frequent contributor to the journal Structure, for which I a m one of the editors. C o n s t r u c t i n g a model from the electron density was in those days h a r d labour a n d very time consuming. For the apoenzyme s t r u c t u r e we had used a contraption called "Fred's folly': invented by Fred Richards d u r i n g a sabbatical with David Phillips in Oxford. It was an optical comparator using a halfsilvered mirror t h a t allowed one to build a physical model into the electron density using Kendrew model c o m p o n e n t s on the scale 2 cm to 1 A (Figure 3). The resulting model was bulky a n d subject to frequent denaturations and distortions. By the time

Fig. 3. The author in front of a model of the enzyme alcohol deh3,drogenase that was built with the aid of a half-silvered mirrol; Fred's folly. The model was built into the mirror image of the computed electron density maps plotted onto large perspex sheets.

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we had the electron density of the holoenzyme, a few groups had started to write programs for virtual model building in specialised computer-display systems developed for flight simulations. The best such program had been written by a postdoc, Alwyn Jones, in the laboratory of Robert Huber in Munich but it was not possible to export the program. During a visit to the lab I managed to convince Alwyn that he should move to Uppsala, be independent and start his own group there. A few months later he came, installed his program on the display in Uppsala and started to export the program to other laboratories. Within a short time this program, FRODO, became the standard modelbuilding program in almost all protein crystallographic laboratories. A novel program called "O" which is also written by Alwyn Jones, is today the standard model-building tool in both industrial and academic crystallographic labs. Alwyn is now a professor at Uppsala University, has a big group in structural biology and is one of my best friends. Protein crystallography in Uppsala made a big leap forward when he joined us.

Thioredoxin and Glutaredoxin Thioredoxin is a small ubiquitous protein that was discovered in Peter Reichard's lab during his work on ribonucleotide reduction. One of his former students, Arne Holmgren, who is now a professor at Karolinska Institutet and a world leader on studies of thioredoxin, suggested in the early 1970s that we collaborate on a structure determination of this protein. I agreed and he started crystallisation studies but all the usual attempts failed. I then suggested adding different metal salts to the crystallisation mixture in the hope that some ions might form links between molecules and promote crystallisation. To our delight this approach succeeded, addition of cupric acetate produced large crystals that diffracted to high resolution. The subsequent structure determination [7] by a PhD student of mine, BengtOlof SSderberg, showed that the cupric ions were linking

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molecules into a layer structure. A similar situation appeared a few years later when we studied glutaredoxin from bacteriophage T4 in collaboration with another former PhD student of Peter Reichard, Britt-Marie SjSberg, who is now a professor at Stockholm University. No crystals could be grown unless cadmium ions were added. The structure determination [8] showed that in this case the cadmium ions linked the protein molecules into chains. I did not pursue studies of these systems any further since I had already started to change my scientific work in other directions, but Hans Eklund continued this collaboration both with Holmgren and SjSberg which resulted in structure determinations of other thioredoxins as well as ribonucleotide reductases.

The Dark Side of Photosynthesis W h e n the ADH project started to approach the point of diminishing returns in the late 1970s, after about 30 publications on the subject, I started to look for more exciting and demanding subjects. I was influenced by the pressure over the years from the Agricultural University to work on something of more relevance for the University t h a n alcohol dehydrogenase and thioredoxins. My argument that the ADH molecule that we were working on came from horse livers did not cut much ice. I knew that I could not expect any collaboration within the University because the projects dominating there were pedestrian and mostly of the type characterised by a friend from the US as "spray and pray". While I was reading up the literature on plant bioscience I came across a report from the National Academy of Sciences in US which was commissioned by President Nixon. The report detailed how modern biological science might be able to contribute to diminish the expected shortage of food worldwide in a long-term perspective. In this report there was a section on the dark reaction of photosynthesis, carbon dioxide fixation, emphasising the low efficiency

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of the key enzyme in the Calvin cycle, ribulose-bis-phosphate carboxylase/oxygenase or Rubisco for short. It was suggested t h a t directed mutagenesis of Rubisco could diminish the oxygenase reaction of this enzyme a n d thereby improve the efficiency of the Calvin cycle by up to 40%. This s o u n d e d to me a worthwhile project a n d I k n e w t h a t a detailed s t r u c t u r e of the p r o t e i n in question was n e e d e d in order to do m e a n i n g f u l sitedirected mutagenesis. I found out t h a t there were several groups already working on the s t r u c t u r e of Rubisco, but they all seemed to have r u n into difficulties so I decided to go ahead. I applied to NFR, where I was the c h a i r m a n for the Chemistry section at t h a t time, for a grant to start this project. The application was t u r n e d down (by the other members of the committee) on the g r o u n d s t h a t the project was too difficult with no prospect of success. The plant enzyme is large with a molecular weight of 500 kDa c o m p r i s i n g eight large a n d eight small subunits. I decided nevertheless to go a h e a d a n d to finance the Rubisco project by using p a r t of my grant for the A D H project, which in their opinion was safe a n d sound. I n e e d e d an experienced biochemist to s t a r t up this project a n d was lucky to be able to recruit Inger A n d e r s s o n who h a d done a very good biochemical work on ADH in Saarbrucken in the group of Michael Zeppezauer. We decided to work on Rubisco from spinach since most of the biochemical work had been done on t h a t enzyme. While Inger was t r y i n g to crystallise t h a t enzyme, which took more t h a n two years, I decided to dirty my h a n d s again on experiments a n d crystallise glycolate oxidase from spinach. This enzyme starts to degrade the p r o d u c t of the oxygenase reaction of Rubisco, glycolate, which only results in heat generation and no storage of energy. I got hold of space in a greenhouse of the University, grew spinach which h a d to be constantly watered, and got sick by spraying poison to kill t h e aphids. I obtained crystals of glycolate oxidase already d u r i n g the latest stage of the purification p r o c e d u r e on the Diaflo filter in the A m i c o n cell used to concentrate the solution. At t h a t stage I was able to recruit Ylva Lindqvist as a P h D

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s t u d e n t to the project who subsequently carried out the struct u r e determination. The u n i t cell was large a n d diffraction was weak, so we realised t h a t we n e e d e d s y n c h r o t r o n radiation to obtain good diffraction d a t a . T h e synchrotron at Daresbury, UK, h a d just come into operation for protein crystallography and the m a n a g e m e n t was willing to sell b e a m t i m e to foreign countries. Together with Ylva's father, who was at t h a t time head of NFR, we a r r a n g e d a contract b e t w e e n N F R and Daresbury which gave b e a m t i m e to Swedish groups after peer review of the projects. We were the first foreign users of b e a m t i m e at Daresbury a n d a picture of Ylva, her h u s b a n d G u n t h e r Schneider a n d I a p p e a r e d on t h e front page of the first issue of Daresbury News. I n this a n d subsequent visits to Daresbury we obtained very good data, Ylva solved the s t r u c t u r e [9] and rapidly became an excellent crystallographer. By this time our group h a d left the chemistry d e p a r t m e n t on the college g r o u n d of the Agricultural University outside Uppsala a n d moved to the newly built Biomedical Center in d o w n t o w n Uppsala. Here we formed our own d e p a r t m e n t of molecular biology which was financed by a special g r a n t from the Agricultural Ministry which t h e Agricultural University reluctantly accepted. We became physically, but not administratively, i n t e g r a t e d w i t h the molecular biology d e p a r t m e n t of Uppsala University to t h e c o n s t e r n a t i o n of a d m i n i s t r a t o r s from b o t h universities. Meanwhile, the Rubisco project h a d t a k e n an u n e x p e c t e d turn. Ylva a n d I had a t t e n d e d the i n t e r n a t i o n a l m e e t i n g on photosynthesis in Greece in 1981 to describe our work a n d to p u t us on the m a p in this field. There we met a n d t a l k e d to George L o r i m e r who worked at the D u P o n t company in Wilmington, US, a n d who was the expert on the biochemistry a n d catalytic m e c h a n i s m of Rubisco. Two years later he p h o n e d me a n d asked if the Rubisco project would be speeded up if the c o m p a n y gave us a grant w i t h no strings attached. Since I still did not have any specific grants for this project, I agreed a n d told h i m t h a t this was an offer I could not refuse. This s t a r t e d a

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very fruitful collaboration not only financially but also scientifically lasting about t e n years and with very enjoyable interactions on the personal level. I n 1984 he told us that a friend of his, Chris Sommerville, h a d cloned, sequenced a n d expressed a bacterial Rubisco which was m u c h smaller comprising only two large subunits. L o r i m e r could provide this enzyme in a purified form provided we were interested in d e t e r m i n i n g the struct u r e as a first step to the plant enzyme. I immediately realised t h a t this was a golden o p p o r t u n i t y a n d over the following years L o r i m e r provided us w i t h m a n y grams of purified enzyme. I m a n a g e d to r e c r u i t G u n t h e r Schneider to be in charge of this project. G u n t h e r h a d come to our lab as a PhD s t u d e n t a n d h a d worked with Hans E k l u n d on metal-substituted ADH gaining m u c h experience in p r o t e i n crystallography a n d h a d just finished his PhD. Within a few m o n t h s he had obtained crystals a n d in 1986 we published the X-ray s t r u c t u r e of this enzyme [10]. This s t r u c t u r e became a t u r n i n g point in t h e studies of Rubisco molecules. Not only did it provide a first picture of the active site of Rubisco, but it also provided the fold of the large subunit of all Rubisco molecules which aided i n t e r p r e t a t i o n of the elect r o n density maps of all the Rubisco molecules that were u n d e r study in different laboratories. Our work on the spinach Rubisco had come to an impasse. All the data were collected but we h a d difficulties in determining the exact heavy-atom positions. I h a d obtained a very bright PhD student, Stefan Knight, who was desperately working with me on this problem. It is a c o m m o n attitude a m o n g noncrystallographers to consider t h a t solving protein s t r u c t u r e s is a trivial p u r s u i t that is nowadays fully automated. In some cases this is correct, but in m a n y cases there are problems that require an intense intellectual effort to solve. In this case we h a d such problems. For two years I struggled with the problem of locating the m a n y heavy-atom positions by a n intellectual p u r s u i t until I realised t h a t computers h a d now become sufficiently fast t h a t b r u t e force m e t h o d s could be applied. Stefan wrote a p r o g r a m which found heavy atoms by these m e t h o d s

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a n d we could calculate an electron density m a p in which we located t h e large subunit c o n t a i n i n g a transition-state analogue b o u n d in the active site. This together with a more detailed view of the bacterial Rubisco provided a w e a l t h of information concerning the geometrical constraints of the catalytic m e c h a n i s m which we published in Nature [11]. However, we could not d e t e r m i n e with confidence the p a t h of the polypeptide chain of t h e small subunit. We n e e d e d to be able to average the density of the four crystallographically indep e n d e n t subunits which were related by a local four-fold axis. However, we now h a d a second tricky problem to solve. The local four-fold axis was almost parallel to a crystallographic two-fold axis, b u t not quite a n d it seemed impossible to determine the exact n a t u r e of deviation. All conventional m e t h o d s failed. It took me several m o n t h s to realise t h a t using the heavy-atom positions we could easily find the exact direction of the four-fold axis. Within a few hours the direction h a d been located a n d an average density was calculated which was easily interpreted. While this was going on a different group t h a t was working on Rubisco from a different plant species h a d published an interpretation of their electron density m a p a n d found a fold for the large subunit very similar to our bacterial Rubisco structure, but they h a d also p r e s e n t e d a s t r u c t u r e for the small subunit. We strongly suspected t h a t their small subu n i t s t r u c t u r e was incorrect since it was incompatible w i t h existing biochemical data. Our spinach Rubisco s t r u c t u r e clearly showed t h a t some connections b e t w e e n secondary struct u r a l elements in their s t r u c t u r e was wrong a n d hence the alignm e n t of the sequence to the fold was in gross error along most of the chain [12]. Shortly afterwards, Alwyn Jones a n d I wrote an article in Nature [13] on errors in published X-ray p r o t e i n structures entitled "Between objectivity a n d subjectivity: We p o i n t e d out t h a t there are aspects of s t r u c t u r e determinations where t h e subjective interpretations of the investigator have consequences for the result a n d therefore objective criteria m u s t be developed a n d used to ensure t h a t the final result is correct. This paper

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has h a d a significant impact. Several criteria have now been developed to test if a s t r u c t u r e might be incorrect. Such criteria will be essential for the high t h r o u g h p u t s t r u c t u r e determinations w i t h i n the s t r u c t u r a l genomic projects. The u l t i m a t e aim of the Rubisco work was not the s t r u c t u r e d e t e r m i n a t i o n of the molecule, b u t t h a t the s t r u c t u r e could serve as a platform for mutagenesis studies t h a t would increase the ratio of carboxylase/oxygenase activity. D u r i n g our structural work we were approached by three biochemical groups, who p u r s u e d such studies for several years on the basis of our s t r u c t u r a l work: Steven Gutteridge in George Lorimers lab, J o h n A n d r e w s from Canberra a n d Fred H a r t m a n from Oak Ridge. It t u r n e d out to be very easy to abolish both activities a n d also to decrease the ratio, but not a single point m u t a t i o n could be found with the desired aim. However, m a n y of these m u t a t i o n s gave significant insights into the role for the carboxylase activity of different residues in the active site. It became apparent after some time t h a t either it was impossible to increase this ratio or t h a t it would require a m u c h more concerted effort to do more sophisticated mutagenesis studies. Sadly enough the g r a n t i n g situation for plant bioscience is such t h a t these efforts were n o t possible to realise a n d I left the field to e m b a r k on a different career, synchrotron m a n a g e m e n t .

A Chaperone for Fibre Formation in Bacteria D u r i n g the Rubisco work I became involved in a newly formed Swedish biotech company, Symbicom. The c o m p a n y h a d been founded by young scientists, one of which h a d sold to a US biotech company, the commercial rights of a gene he h a d discovered for a price t h a t was more t h a n a t h o u s a n d times less t h a n the profit it soon generated yearly to the company. The focus of Symbicom was on p r o t e i n - c a r b o h y d r a t e interactions a n d comprised three labs in the areas of bacterial molecular genetics, carbohydrate chemistry a n d m a m m a l i a n carbohydrate surface

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antigens. The c o m p a n y w a n t e d a f o u r t h leg in protein s t r u c t u r e d e t e r m i n a t i o n a n d c o m p u t e r modelling a n d I agreed to set u p a n d m a n a g e such a lab in Uppsala in my spare time. I was able to r e c r u i t good scientists to this lab w i t h an excellent m a n a g e r who soon r a n the lab with a very little intervention from my side. Symbicom was sold in 1994 to the big pharmaceutical company, Astra which is now p a r t of Astra-Zeneca. Symbicom was a typical early biotech company t h a t was r u n by scientists more interested in science t h a n in commercial exploitation. One of the founders, Staffan N o r m a r k from Ume~, was working on a bacterial operon t h a t codes for the proteins involved in the formation of pili, fibres t h a t initiates u r i n a r y infection by a t t a c h i n g the bacteria to carbohydrates on the surface of m a m m a l i a n cells in the u r i n a r y tract. One of these proteins, PapD, was responsible for t r a n s p o r t i n g the fibre subunits in the periplasmic space to the site of pili assembly in the outer membrane. We were excited about the prospect t h a t a s t r u c t u r e d e t e r m i n a t i o n of PapD in complex w i t h a fibre subu n i t might reveal details of the assembly mechanism, so we s t a r t e d a scientific collaboration in this area. We b o t h agreed t h a t h a d it not been for our m u t u a l e n g a g e m e n t in this c o m p a n y we would probably never have s t a r t e d this exciting scientific project. I r e c r u i t e d a PhD student, Anders Holmgren, who h a d experience of p r o t e i n expression, to express, purify, crystallise a n d d e t e r m i n e the s t r u c t u r e of PapD alone which seemed to me a suitable PhD project in those days. The project went smoothly along a n d in 1989 he p r o d u c e d an electron density m a p which was reasonably easy to interpret. W h e n I looked at his prelimin a r y chain tracing I immediately saw t h a t by c h a n g i n g a few connections the fold of b o t h domains became typical immunoglobulin domains. Subsequent refinement confirmed my subjective interpretation. The discovery of an i m m u n o g l o b u l i n d o m a i n in a bacterial protein h a d not been made before. I immediately s t a r t e d to look for evidence of horizontal gene transfer from the k n o w n sequences of m a m m a l i a n immunoglobulin-like domains

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but could not find any significant sequence homology, only borderline sequence similarities. However, my frequent interactions on the Rubisco project with George Lorimer and John Ellis, who were at that time heavily engaged in GroEL, had made me aware of the importance of the chaperone concept and I realised that what we saw in the PapD structure was a chaperone that prevented the fibre subunits from improper assembly in the periplasmic space. We composed a letter to Nature entitled "Crystal structure of a chaperon protein, PapD, reveals an immunoglobulin fold" and rapidly obtained a reply saying that they would be happy to accept the paper, but could I please extend it to a full article [14]. Such a response from Nature is probably a rare experience in the life of a scientist. Staffan Normark had at that time moved from Ume~ to greener pastures as c h a i r m a n of the prestigious department of microbiology at Washington State University in St. Louis. The pill project had been taken over by one of his postdocs, Scott Hultgren, who had moved with Staffan to St. Louis. I continued the mutually fruitful collaboration with Scott to explore the implications of the PapD structure and to continue the X-ray work focussed on PapD complexes. The X-ray work was eventually taken over by Stefan Knight who after many years of struggle with bad crystals managed to obtain a structure of a complex with PapD and from this structure to deduce a very plausible mechanism of pill fibre assembly, an excellent piece of scientific deduction.

Introduction to Protein Structure: A Textbook

W h e n protein engineering started to become a common tool in biochemistry and molecular biology labs, I realised that protein structure determination would rapidly increase in importance and be transformed from an esoteric academic pursuit to an important aspect of molecular life science. Without an understanding of the principles of protein structure, protein

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e n g i n e e r i n g degenerates into tinkering. I also realised by reading a n u m b e r of papers t h a t the knowledge of the principles of protein s t r u c t u r e was a scarce c o m m o d i t y a m o n g molecular biologists a n d t h a t m a n y s t r u c t u r a l biologists were r a t h e r ignora n t of the work from laboratories other t h a n their own. These were some of the concerns t h a t made me t h i n k of w r i t i n g a textbook on protein s t r u c t u r e s in the mid-1980s. There was no m o d e r n textbook on this subject which was one of t h e reasons t h a t there were only a few university courses on protein structure. The only way to b r e a k this vicious circle was to write a textbook a n d hope t h a t some publishing c o m p a n y would be brave enough to publish it despite the negative recommendations from their m a r k e t i n g department. In 1987, the urge to write such a b o o k h a d become an obsession for me. I realised t h a t I n e e d e d a coauthor, not in s t r u c t u r a l biology which I mastered quite well, b u t someone who would represent the t a r g e t scientist a n d be a clever a n d intelligent biologist w i t h little knowledge of s t r u c t u r e b u t w i t h experience of w r i t i n g books for biologists. I k n e w the person I w a n t e d as coauthor, J o h n Tooze, M r EMBO, who had r u n the E u r o p e a n Molecular Biology Organization almost from its inception, who had writt e n several books in molecular biology a n d w h o m I k n e w a n d respected from m a n y years of interaction in EMBO committees. The EMBO office was located in the premises of EMBL, the E u r o p e a n Molecular Biology Laboratory in Heidelberg, the Director General of which was an old friend of mine, L e n n a r t Philipson from Sweden. He accepted me as a visitor for six m o n t h s a n d I left my lab a n d my family to go there on a sabbatical to write the book. W h e n I came a n d confronted J o h n Tooze with my plans he was aghast. I hate s t r u c t u r e s he said, b u t since you are here give me a chapter to look at a n d I will decide if I a m with you or not. W h e n I gave h i m the chapter on DNA-binding proteins, he became as excited as J o h n can show a n d told me: "Not bad, b u t it has a lot of jargon a n d u n n e c e s s a r y s t r u c t u r a l details which I already have changed". T h a t s t a r t e d our excellent a n d

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very fruitful collaboration. We quickly agreed t h a t the book should not have any stereo pictures b u t instead an a b u n d a n c e of colour pictures in mono, each of which would highlight only one aspect of the structure. J o h n contacted several of the large publishing companies but none of t h e m was willing to publish a book t h a t required colour on every page, a cheap price a n d t h a t had no university courses on the subject. Finally he got in contact with Gavin Borden, the owner of a small US publishing company, Garland, whose only book in science was "Molecular Biology of the Cell", which he h a d published in similar circumstances on the advice of Bruce Alberts. Gavin accepted to take the chance to publish our book a n d agreed to all our conditions. After six m o n t h s of intensive work in Heidelberg, J o h n a n d I h a d w r i t t e n h a l f the book. My family was complaining, the lab was at a standstill a n d I had to r e t u r n to Uppsala. It took us t h r e e years to complete t h e book including the frequent interactions with the staff at the Garland office in London; t h e skilled text editor, M i r a n d a Robertsson, a former Nature biology editor who became a great friend of mine, the picture editor Keith Roberts and the designer Nigel Orme who converted my sketches to beautiful figures in the book. "Introduction to P r o t e i n Structure" [15] became a great success a n d has been read by more t h a n one h u n d r e d t h o u s a n d students a n d scientists, according to a c o m p a n y survey. It still gives me enormous pleasure to receive letters or to meet u n k n o w n scientists informing me how m u c h they have appreciated the book and t h a t it has h a d a s t r o n g influence on their science. Being the a u t h o r of a textbook can have m a n y interesting repercussions. Some years ago I was approached by a rising star in n a n o t e c h n o l o g y of biomaterials, S h u g u a n g Zhang, at MIT. He claimed t h a t "Introduction to P r o t e i n Structure" had been very i m p o r t a n t to give h i m new ideas in his design of peptides useful for nanomaterials. He had originally left China to study Z-DNA with Alex Rich because he was fascinated by the left-handedness of the Z-DNA helix. His studies of the sequence of a Z-DNA-binding p r o t e i n diverted h i m into design of peptides

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that could self-assemble into fibres or sheets that are useful as biomaterials. Over the years we have become close friends. I have often wondered how an organisation that funds research in nanomaterials would react to an application from an u n k n o w n Chinese student who wanted to study left-handed helices in the US. They would probably t u r n his application down and miss funding one of the most interesting developments in biomaterials on a nanoscale. Quite recently I obtained an email from a Canadian artist, Jacques Deshaies, who has specialised in making artistic paintings based on DNA and chromosomes [16]. He has made several front covers of Nature Genetics and other journals. He told me that he had recently started to make paintings based on protein structures, had been recommended "Introduction to Protein Structure" and became inspired to convert the scientific illustrations in the book into artistic paintings. A few months later he had an exhibition in Uppsala on his latest proteome paintings and we had a very interesting evening together. To my knowledge he is the first artist who has used protein structures as a theme for an exhibition and I am proud that our book inspired him to do that.

Structural Biology at Synchrotrons In 1991 I was offered the job as research director of the European Synchrotron Research Facility, ESRF, which was being built in Grenoble, France, as a joint venture by 12 European countries. This facility was going to be the flagship of European synchrotron research, the first 3rd generation highenergy machine which produced X-ray beams from insertion devices that were about eight orders of magnitude more brilliant t h a n those from existing synchrotrons. The machine was by that time in the final stages of construction under the leadership of the brilliant machine physicist Jean-Louis Laclare who unfortunately passed away in April 2003 after a long period of illness. A few beamlines with experimental stations mainly for

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experiments in physics were in the planning stage. My job would be to manage the build-up of experimental facilities in biology, medicine and chemistry for the use of thousands of scientists from the member countries. I realised that this facility was a golden opportunity for extending the frontiers of structural biology by allowing structure determinations of very large molecular complexes as well as collecting data from very small crystals. In addition, it might be possible to study the dynamics of biochemical reactions by time-resolved crystallography in the microsecond range, obtain fibre diffraction patterns of single fibrils and explore novel medical applications [17]. Furthermore, I had the vision to make ESRF the focal point for structural biology in southern Europe which was at that time weak but where there were an abundance of excellent biochemists and molecular biologists, many of whom I knew from my involvement in EMBO and EMBL. In those days the idea of making biological research one of the major activities of a synchrotron radiation facility was not popular among synchrotron physicists who wanted these precious facilities for their own research. To my surprise I was offered the job immediately after the job interview during which I made it quite clear that I would only take the job if I had support for my ideas. Full of enthusiasm to realise my plans I arrived at ESRF in April 1992 to be immediately thrown into an unexpected, chaotic and almost Kafka-like situation. The floor of the 16,000 square metre experimental hall had started to crack and pieces of the floor were wobbling several m m when walked upon. The alignment of the X-ray beams along the beamlines could only tolerate vibrations up to 10 pm. My codirector for research in physics, Massimo Altarelli, had decided that no beamlines should be built until the floor was repaired or rebuilt. The machine was in operation ahead of the time schedule producing an X-ray beam of unparalleled quality far above the original specifications. The physicists that had built this machine wanted of course this beam to be used as soon as possible for exciting and novel experiments. For some reasons the director

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general, Ruprecht Haensel, sided wholeheartedly with the machine physicists. I required only a few days to agree with Altarelli that the floor was unsuitable for beamlines. The management thus became split into two groups which were in violent conflict for several months about the floor situation. Meanwhile, Altarelli who is a theoretical physicist, became deeply involved in the mysteries of concrete by consulting a number of experts on how to repair the floor. In other circumstances it would have been amusing to see how widely the expensive experts differed in their advice. The director general tried to solve the floor problem in his own way by sending Altarelli and me a formal letter to the effect that we were fired from our jobs since we had not obeyed his order to start building beamlines on the existing floor. To his surprise the c h a i r m a n of the ESRF Council, Jules Horowitz, informed us that we could ignore the letter since only he could hire or fire directors. A few months later we had a new director general, Yves Petroff, the floor was repaired by filling the voids under the concrete plates with grout, and an intense period followed with construction of the beamlines that had been planned. In a subsequent lawsuit against the company that had built the floor, ESRF recovered all extra costs including compensation for the delay in operation of the facility. Structural biology was virtually non-existent at ESRF when I arrived. Fortunately, EMBL had an outstation in Grenoble which originally had been created in order to provide experimental facilities for structural biology at the neutron source, ILL. Since structural biologists rarely use neutron radiation, this outstation had been small and did not take any significant part in the development of structural biology in Europe. In the mid-1980s the new director of this outstation, Stephen Cusack, started to build-up a strong inhouse group in X-ray macromolecular crystallography which was now ready to play an active part in the development of structural biology at ESRF. A full collaboration was, however, delayed due to differences in internal policy of the councils of the two organisations, EMBL

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and ESRF. Pragmatic decisions are sometimes difficult to implement in bodies governed by different European countries. In the end I managed to obtain support for the establishment of a joint collaborative group which formed the foundation for the very successful joint structural biology and structural genomic efforts which are now taking place in Grenoble. Stephen Cusack's engagement in t h e synchrotron coupled with financial support from the EMBL budget was crucial for building up state-of-the-art beamlines for structural biology at ESRF. The strong support that we obtained from Yves Petroff was essential for overcoming the reluctance from some of the physicists in the Council and at ESRF to divert resources from real physics to structural biology. Gradually, a warm friendship developed between Yves and myself, which also included our wives, and it was always a privilege to enjoy the delicious meals cooked by Yves himself. The use of synchrotron radiation in structural biology underwent a rather dramatic change during my time at ESRF, from 1992 to 1997. Until 1992 the phase information that is required to solve a novel structure was almost exclusively obtained by the method of isomorphous replacement that Max Perutz had developed in the 1950s. By 1997 this method had been essentially replaced by a different approach requiring synchrotron radiation. It had been known for a long time that the anomalous diffraction signals caused by irradiating an atom in a crystal by X-rays with an energy in the vicinity of its X-ray absorption edge contained information that could be used to determine the phase angles of the diffraction pattern of the crystal. However, practical application of this method was hampered by the lack of sufficiently stable X-ray beams of variable energy and difficulties in measuring the small anomalous signals with sufficient accuracy. In addition, the method required the presence of one or more intrinsic heavy atoms in the macromolecule. Pioneering studies by Wayne Hendrickson to produce more stable synchrotron beamlines and to apply recombinant DNA technology to produce proteins containing seleno-methionine solved these

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problems. Today more t h a n 90% of all novel structures are determined using anomalous diffraction at synchrotron beamlines to determine the phase angles. The combined efforts of ESRF and EMBL in Grenoble played a significant role in this later development. We all became excited when we realised from the very first experiments that ESRF heralded a new era for synchrotron research. The properties of the X-ray beam from this 3rd generation synchrotron source t u r n e d out to be ideal for structural biology. Data could be collected with orders of magnitude more rapidly, very large molecules could be studied (Figure 4) and data could be obtained from crystals that were too small to give useful diffraction from any other X-ray source. In addition, the position of the beam was so stable that successful MAD . . . . ~ 4 84:

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X-ray diffraction pattern of a crystal of blue tor~gue virus recorded at one of the beamlines of E S R F equipped with an undulator insertion device. The unit-cell dimensions of these crystals are around 1000A units and it requires a very parallel beam to be able to separate the diffraction spots. The photograph was taken by David Stuart and his group at Oxford University. F i g . 4.

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experiments became a routine operation. W h e n eventually commercial CCD detectors became available the speed of data collection increased a n o t h e r order of magnitude. Today, a complete dataset can be obtained in about an hour c o m p a r e d to several days t e n years ago. W h e n I came to ESRF there was one experimental station p l a n n e d for macromolecular crystallography, w h e n I left in 1997 there were six in operation or construction a n d today (2003) there are about ten. Worldwide there were about 15 such stations in operation in 1992, today there are about 60 a n d a n o t h e r 40 are financed to be built, m a i n l y at the new 3rd generation synchrotron sources now u n d e r construction. This rapid increase was to some extent initiated by the success of ESRF a n d has recently been fuelled by the large a m o u n t of funds for S t r u c t u r a l Genomics. There was never a dull m o m e n t in the life at ESRF d u r i n g these years. We were engaged in an ambitious p r o g r a m to build a n d operate beamlines to be used by physicists, chemists and biologists. ESRF was pioneering the use of such strong X-ray beams a n d m a n y new inventions had to be developed and tested. There were endless meetings and negotiations with representatives from the twelve-member countries as well as scientists from these countries who w a n t e d to build facilities for their pet projects. On the bright side we were able to recruit a n u m b e r of brilliant scientists who were attracted by the novel scientific opportunities created by this X-ray source. The rapid influx of scientists a n d engineers with different cultural backgrounds p r e s e n t e d a range of interesting problems for the management. I soon adopted the motto of Yves Petroff: "The role of a Director is to solve problems, not to create them': Selection of external users of b e a m t i m e created special problems due to the conflict between the highest scientific merit of the projects a n d "juste retour': t h a t is allocation of b e a m t i m e to scientists from individual countries in proportion to the a m o u n t of money the country contributed to the budget of ESRF. Creative bookkeeping solved some of those problems, but those council members who were civil servants were always more interested in "juste

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retour" than in high scientific standards. However, the large number of articles based on data collected at ESRF that were published in Phys. Rev. Letters, Nature and Science during these years testify both to the success of selection based on high scientific merit and the high scientific quality of the staff scientists and external users in such diverse fields as material science, geochemistry, basic physics and structural biology. My own time for research was extremely limited during this time. However, I managed to initiate studies on single silk fibres from spiders [17] using a microfocus beamline that had been constructed by Christian Riekel. He has since continued these studies and obtained remarkable results. I remarried a few weeks before leaving Sweden for France. My new wife, Malin Akerblom, works in an organisation within Uppsala University that supports basic-science projects in the poorest countries of the Third World such as Tanzania, Zimbabwe, Sri Lanka and Bangladesh. Through her and her work I have had the opportunity to obtain a close insight into the problems that face capacity building in science in these countries, but I have also had the privilege of getting to know a number of bright and devoted Third World scientists who struggle hard against difficult odds. From a global perspective of science policy it is a dreadful waste of human resources that the industrialised world supports mediocre scientists in large numbers, while many bright young people in the Third World are deprived of the possibility to make their impact on the development of science.

Science Policy and Advice A significant fraction of my time has been spent in committees, both national and international. The intellectually most rewarding of these activities has been to serve for nine years on the Nobel Committee for Chemistry, the body which selects and recommends the Nobel Prize winner(s) in chemistry for decision

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by the Royal Swedish Academy of Science. It is a small committee, comprising five ordinary members, one secretary a n d two to t h r e e co-opted individuals. Each year the c o m m i t t e e sends individual invitations to t h o u s a n d s of members of academies, university professors a n d other scientists in n u m e r o u s countries asking t h e m to n o m i n a t e candidates for the Nobel Prize for the coming year. The nominations received are t h e n evaluated by the c o m m i t t e e w i t h the help of domestic and foreign experts. These experts are invaluable for an objective selection of candidates t h a t are p u t on a shortlist, t h a t is those t h a t are considered worthy of obtaining a Nobel Prize. The selection of the winner(s) from the shortlist is more difficult a n d occasionally somewhat subjective, since everyone on the shortlist could in principle be selected. The evaluation p r o c e d u r e requires a considerable a m o u n t of work by the c o m m i t t e e members, since several h u n d r e d different candidates are n o m i n a t e d each year. On the other hand, it was a very r e w a r d i n g activity since I was exposed to virtually all scientific frontiers in chemistry a n d obtained a n u m b e r of friends a m o n g the experts we consulted as well as a m o n g the prize winners themselves. A n additional benefit is the ready acceptance of possible candidates to visit Sweden a n d lecture at symposia or seminars, especially if these are held u n d e r the auspices of the Nobel Foundation. D u r i n g the 1980s a n d 1990s I served in a large n u m b e r of f u n d i n g agencies a n d science advisory committees, such as Science Research Councils in Sweden, the EMBO F u n d C o m m i t t e e for postdoctoral fellowships, the Fachbeirat of Max Planck Institutes a n d scientific advisory committees of several s y n c h r o t r o n facilities. Based on my own experience as a young scientist my m a i n concern in these committees was to support young scientists who w a n t e d to break away from their supervisor and enter a novel field of research. Too m a n y times I failed because such an action usually involved diversion of funds to risky projects from older scientists with s o u n d and safe projects. Most c o m m i t t e e members objected to such actions.

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The most interesting of these committees from the human point of view was the Science Advisory Council of the Swedish Government. This council was chaired by the prime minister Ingvar Carlsson who attended almost all the monthly meetings together with several other ministers. It is rather unusual that the governments pay such strong attention to science matters and the result has been that Swedish scientists have had a strong influence on science policy matters and have been able to promote political decisions that have been favourable for science and prevent those that would have been harmful. Each meeting was devoted to one specific subject, suggested and organised by one of the committee members. The chairman of the council, Bert Bolin, managed to obtain support from the Government for the Global Change program with headquarters in Sweden and with strong initial financial support from Sweden. This program has been instrumental for scientific studies on environmental changes on a global scale that has been caused by human activities.

Epilogue When I left Sweden for France in 1992 1 resigned from my chair in molecular biology at the Agricultural University. I realised that after my five-year period at ESRF I would have two years left without a job before I retired, but I convinced myself that something would pop up. It did. My old friend from the Symbicom time, Staffan Normark, who had just returned to Sweden from US and taken up a position at the Karolinska Institute in Stockholm told me to apply for a medical Nobel institute guest fellowship for my remaining two years. I applied, obtained the fellowship, and was given office space and a warm welcome by the chairman of the Microbiology and Tumorbiology Center, MTC, at the Karolinska Institute, Ingemar Ernberg. The conditions for the fellowship suited me perfectly. I was not allowed to take on any teaching or administrative duties,

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instead I should use the time for my own scientific or scholarly work. The second edition of "Introduction to P r o t e i n Structure" was long overdue, there had been very little time in Grenoble to work on this book. I realised t h a t so m u c h h a d h a p p e n e d in s t r u c t u r a l biology d u r i n g the last decade t h a t a simple u p d a t e of the book would not be sufficient. By the time I h a d finished, more t h a n one year later, I h a d completely r e w r i t t e n more t h a n h a l f the book [19]. However, I did not change the concept of the book which was to write for biologists a n d not for biophysicists. One consequence of that concept is t h a t not a single mathematical equation is present in the book with the exception of Bragg's law for diffraction in the last chapter. M a n y biologists have expressed their delight about this concept a n d m a n y biophysicists have complained. Since the book is m a i n l y used as a textbook for courses in biophysics I have realised t h a t an entirely different concept is now required a n d I have been f o r t u n a t e enough to join J o h n Kuriyan to write a new book in S t r u c t u r a l Biology that is more biophysically oriented. In 1993 1 was asked by Wayne Hendrickson if I w a n t e d to join h i m as an editor of a new journal devoted to s t r u c t u r a l biology t h a t C u r r e n t Biology was interested in publishing. A few years earlier, when I was the c h a i r m a n of the Macromolecular Commission of the I n t e r n a t i o n a l Union of Crystallography, I h a d tried to interest Acta Crystallographica in s t a r t i n g a special journal for macromolecular s t r u c t u r e s b u t w i t h no success. As late as 1993 there was still no journal devoted to s t r u c t u r a l biology, so t h e c o m m u n i t y published their s t r u c t u r e s in a bewildering variety of journals. I was therefore immediately interested in Wayne's suggestion a n d we met at C u r r e n t Biology with Peter Newmark, a former Nature biology editor, to discuss the terms. The journal, Structure, s t a r t e d in September 1993, a n d by the time I left Grenoble in 1997 the field h a d grown exponentially a n d Nature S t r u c t u r a l Biology h a d become our m a i n competitor. Already at the b e g i n n i n g we obtained m a n y more m a n u s c r i p t s t h a n we were allowed to publish which p u t an extra b u r d e n on the editors. We read all the m a n u s c r i p t s a n d

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made a first decision if we should reject it or send it to referees. A l m o s t all the m a n u s c r i p t s were technically sound a n d of interest to at least a p a r t of the scientific community, but we h a d to m a k e a choice based on novel biological insight of the work reported. In 1993 the field was still sufficiently small so t h a t I knew most of the groups a n d their work in s t r u c t u r a l biology a n d could assign referees. As the years went by I spent more a n d more time reading m a n u s c r i p t s and finding suitable referees. On the positive side I was forced to closely follow this rapidly e x p a n d i n g field, which was very useful for me b o t h as a science advisor and for w r i t i n g the books. At the age of 65 a Swedish scientist is supposed to cease all her scientific activities a n d retire to a quiet life on a reasonable pension. The research councils give no support to a scientist above 65 years of age and she is not allowed to supervise any PhD student. Since I h a d been involved in b r i n g i n g about these rules, I h a d been p r e p a r e d for this d u r i n g the two years between r e t u r n i n g from France and my retirement. I h a d used my fellowship to learn bioinformatics a n d had participated in some research projects led by scientists at MTC [20]. I was looking forward to a quiet life with time to immerse myself in bioinformatic studies of genome sequences combined with protein structures. I was very pleased t h a t MTC w a n t e d me to stay on as a professor emeritus and participate in an a t t e m p t to build-up bioinformatics in an essentially cell biology environment [21]. However, almost from the b e g i n n i n g of my life as a retired scientist I was asked by a n u m b e r of organisations to participate in various committees. I was too flattered to realise that vanity is the enemy of science, so I accepted most of these requests a n d found myself s p e n d i n g more a n d more time on airplanes flying from one s y n c h r o t r o n facility to a n o t h e r or from one large Japanese, US or E u r o p e a n I n s t i t u t e to a n o t h e r evaluating their s t r u c t u r a l biology efforts. In addition, I was involved in a n u m b e r of science policy a n d f u n d i n g activities in Sweden. This hectic lifestyle abruptly came to an end by November 2002 when by accident it was discovered t h a t I h a d developed

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lung cancer a n d I immediately resigned from all my external activities a n d s t a r t e d t r e a t m e n t in the form of b o t h chemotherapy a n d radiation therapy. The c h e m o t h e r a p y so far has been r a t h e r conventional based on taxol a n d a cis-platinum compound. The prognosis for lung cancer is still quite bad, especially w h e n the big p r i m a r y t u m o u r is accompanied by a n u m b e r of small secondary t u m o u r s in b o t h lungs. From a h u m a n point of view it has been a fascinating experience to suddenly realise t h a t the end of my life is in sight. Looking back, not least by w r i t i n g this essay, I find great comfort in the fact that I have h a d a full a n d rich life with m a n y friends all a r o u n d the world, been blessed with children a n d grandchildren a n d never h a d a boring moment, t h a n k s to my scientific activities. I have decided that since I have devoted almost my entire life to science I also want to contribute to science towards the end of my life by p a r t i c i p a t i n g in clinical trials based on novel c o m p o u n d s such as protein kinase inhibitors. I feel confident that t e n years from now lung cancer will be regarded as a curable disease, t h a n k s to the steady advancement of our knowledge of the molecular biology of cancer.

ACKNOWLEDGMENTS I wish to express my sincere t h a n k s to Professor Julia Hasler at t h e University of Zimbabwe, Harare, for her excellent linguistic corrections of this essay.

EDITOR'S FOOTNOTE Carl-Ivar Br~ind~n passed away on April 28, 2004, two weeks short of his 70th birthday (on which occasion he would have received the h o n o r a r y doctor degree from the Karolinska Institutet). W h e n he s u b m i t t e d to us his autobiographic chapter he k n e w t h a t he h a d a lung cancer in an advanced stage. I could

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but a d m i r e at the way he looked at his disease as a novel, enriching experience; t h e r e a d e r is never asked to s h a r e w h a t e v e r a n x i e t y or feelings of a p p r o a c h i n g a n u n t i m e l y farewell he m a y have had, but r a t h e r to s h a r e his love for nature, p a r t i c u l a r l y t h a t of "his" Lappland, a n d his f a s c i n a t i o n for p r o t e i n crystallography a n d its c o n t r i b u t i o n to enzymology. Carl's colleagues a n d friends have w r i t t e n obituaries (e.g. in the Svenska Dagbladet of M a y 17th a n d of J u n e 2nd, a n d in Nature, Structural & Molecular Biology, J u n e 2004, vol. 11, pp. 490-492). F r o m t h e last-mentioned o b i t u a r y I quote w h a t Eva Klein (the r e n o w n e d i m m u n o l o g i s t at t h e Karolinska) once r e m a r k e d : "I have always t r i e d to find a g r e a t scientist, who is genuine, gentle, modest, h o n e s t a n d accessible. I have found t h e person, Carl Br~ind~n". Eva Klein expressed in a perfect m a n n e r the feelings of all of us who h a d the luck of g e t t i n g to k n o w Carl.

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