14th Course : Crystallography of Molecular Biology

29 May to 7 June 1988


Directors : Wim J. G. Hol, Groningen

Purpose of the Course

It is more than seventy years since the discovery of the X ray diffraction by crystals, and crystallography has since established an ever widening sphere of influence in science. The solving of crystal structure has had an increasing impact in many fields - for example physics, chemistry, biology, materials science, medicine, metallurgy, mineralogy and geology.

Postgraduate courses at the "Ettore Majorana" Centre for Scientific Culture periodically review the advancement of crystallography. They usually concentrate onm interdisciplinary frontier topics.

The purpose of this Course is to discuss new techniques and review recent developments in the crystallography of molecular biology with special emphasis on proteins, nucleic acids and viruses. New techniques include those which have become available through the introduction of new technologies - synchrotron radiation, raster graphics, supercomputers, area detectors, crystallization in space. New theoretical approaches include maximum entropy methods, relational data base searches, density modification and phase extension. Crystallization of biomacromolecules will be given ample attention because of its crucial role in structure determination projects. The Course will stress the principles of folding and dynamics of DNA, RNA and proteins. The process of membrane passage by proteins will be related to structural features. Potential applications will be given by focusing on protein engineering, rational drug design and development of vaccines.
A central purpose of the course will be that of promoting mutual understanding and possible future collaboration.

Topics (scientific sequence) and LECTURERS (alphabetic)

Crystallization of biomacromolecules T.L. BLUNDELL, Birbeck College, London, UK
Laue methods C.J. BRANDEN, Uppasala University, Sweden
Electronic area-detectors G. BRICOGNE, University of Paris, France
Synchrotron radiation P. COLMAN, CSIRO, Parville, Australia
Phase determination methods R. GIEGE', Universitè Strasbourg, France
Dynamic crystallography W.A. HENDRICKSON, Columbia University, NY, USA
Computer graphics O. HERZBERG, University of Alberta, Edmonton, Canada
Enzyme catalysis A. HOWARD, Genex, Gaithersburg, USA
Protein-antibody interactions R. HUBER, Max-Planck Institute, Munich, F.R. Germany
Structure of RNA's J. N. JANSONIUS, Biozentrum, Basel, Switzerland
Membrane proteins L.N. JOHNSON, University of Oxford, UK
Chromatin and nucleosomes A.T. JONES, Biomedical Centre, Uppsala, Sweden
Viruses A. KOSSIAKOFF, Genentech, San Francisco, CA, USA
DNA binding proteins D.A. MATTHEWS, Agouron Institute, San Diego, CA, USA
Bacterial toxins M. F. PERUTZ, MRC, Cambridge, UK
Ribosomes G. PETSKO, MIT, Boston, USA
Mobility of proteins R.J. POLJAK, Institut Pasteur, Paris, France
Structure of DNA M.G. ROSSMANN, Purdue University, West Lafayette, IN, USA
Glycoproteins G. SCHATZ, Biozentrum, Basel, Switzerland
Molecular modelling T.A. STEITZ, Yale University, New Haven, USA
Rational drug design D. SUCK, EMBL, Heidelberg, F.R. Germany
Protein engineering B.K. VAINSHTEIN, Moscow, USSR
Protein membrane passage W.F. VAN GUNSTEREN, Groningen University, Netherlands
  A. WANG, MIT, Boston, MA, USA
  B:C: WANG, University of Pittsburgh, PA, USA
  D.C. WILEY, Harvard University, Cambridge, USA
  K. WILSON, EMBL, Hamburg, F.R. Germany
  A. YONATH, Weizmann Institute, Rehovot, Israel


A Report

The course was well attended by about 210 participants from 36 different countries and covered a wide spectrum of crystallographic investigations devoted to understanding the structure and function of biomacromolecules such as DNA, RNA, viruses and ribosomes. New developments in techniques to determine structures were also covered, as were applications or potential applications in the fields of rational drug design and protein engineering.

A total of 60 lectures were given which were, virtually without exception, of excellent quality indeed. The discussion period after each lecture was a nice forum for exchange of opinions enthusiastically used by the participants. Also the poster sessions were very lively and sparked off many discussions. But not only the quality of the lectures, also the perfect local organization and kindness of the organizational team contributed in a major way to a most successful meeting. On top of all that, the setting of Erice - a small town situated on a hill - plus ideal "conference weather" did help in ensuring a virtual one hundred per cent attendance up to the very last contribution.

On the first day new developments in crystallization techniques were covered by Richard Giegè, Strasbourg, making clear, however, that trial-and-error is still a major component of growing good quality crystals of biomacromolecules. Joel Sussman, Rehovot, described a major improvement in extending the lifetime of crystals in the x-ray beam: "shock cooling" by sudden exposure to a cold nitrogen gas stream, or to liquid butane. It is the intention of the European Science Foundation Network of Crystallography of Biological Important Molecules to devote a special practical course to this new technique as soon as possible so that it becomes well established in the European Community.

In the next session David Matthews, San Diego, described how the binding of anti-bacterial drugs to a target enzyme is employed for designing new anti-bacterial compounds and reported about the recently determined structure of thymidylate synthase. Rik Wierenga, Groningen, described the progress of a quite ambitious project aimed at the design of of new sleeping sickness drugs. Despite very small amounts of protein available considerable progress i.e. unravelling the structure of the first two enzymes from a tropical parasite, had already been made.

Robert Poljak, Paris, and Peter Colman, Australia, described fascinating results of antibodies complexes with lysozyme and influenza virus neuraminidase, respectively. Luhua Lai, China, reported on modelling a structure of a protein inhibitor by molecular dynamics techniques. While the first day was concluded by Alwyn Jones, Uppsala, showing how a data base of protein structure fragments is of tremendous help in building structures of new proteins.

The DNA session of the second day gave a firm foundation of principles by Andy Wang, MIT, then saw the binding of spermine in the major groove od DNA by Sundaralingam, Madison and ended with an example of looped-out base to accommodate a mis-match by Joel Sussman, Rehovot. DNA-protein interactions were shown in magnificent detail in the next three lectures where the bending of DNA to interact with quite rigid protein molecules like trp and 434 repressors became evident. After poster session I, the second day was concluded by Robert Huber, Muenchen, describing most impressive results on light energy harvesting protein complexes and by Jeff Schatz, Basel, who presented a splendid lecture showing how protein molecules reach their proper destinations in the cell.

The third day contained the synchrotron session in which Keith Wilson, Hamburg, described basics to the uninitiated and mentioned the imaging plate. Mitchell Guss, Australia, explained the use of the multi wavelength method in solving the phase problem of a small metalloprotein. Perhaps the most revolutionary talk of th meeting was given by Janos Hadju, Oxford, who showed tremendous progress made in data collection by the Laue method and predicted that in the near future "single bunches" of electrons in a synchrotron would be sufficient to record diffraction patterns within +/- 100 pico-seconds. The result on a virus structure were most impressive. Also here, the ESF Network is seeking means to allow young European scientists to obtain "hands-on" experience with this method by organizing a workshop. The third day saw also two excellent lectures on the catalysis by transaminases by Hans Jansonius, Basel and on triose phosphate isomerase by Greg Petsko, MIT.

The fourth day started wih principles, power and pitfalls of the molecular dynamics techniques by Wilfred Van Gunsteren, Groningen, followed by a lucid account of catalysis by phosphorilase, a very large and well controlled enzyme.(Louise Johnson, Oxford). Gerard Bricogne, Paris, described developments in in a statistical theory of the phase problem aiming to incorporate all possible sources of phase information into one unified field theory. Bi Chen Wang, Pittsburgh, gave a very practical account of the solvent flattening and automatic envelope determination procedures developed in his laboratory. An important point appeared to include calculated structure factors in cases where structure factors were missing.

The refinement session included some beautiful examples of the recently developed molecular dynamics refinement technique. Piet Gros, Groningen, showed how the radius of convergence of this new procedure us dramatically increased compared with conventional refinement methods, while Tom Garrett, Harvard, gave impressive examples of the improvement of the electron density distribution even in places where atoms had not automatically moved towards their correct position. This cleaning up of the electron density is obviously of tremendous importance during rebuilding session on graphics systems.

The virus session of the fifth day contained three splendid contributions. First, Michael Rossmann, Purdue, described the detailed insight obtained in the binding of a series of compounds to rhinovirus, which prevent uncoating of the viral particle and may be the starting point of a new generation of anti-viral drugs. Then Cynthia Stauffacher, Purdue, described with great enthusiasm the first example of a RNA bound to the inside of an icosahedral virus. It appeared to have a conformation quite similar to that of a loop in tRNA. Gerald Stubbs, Nashville, described RNA binding to Tobacco Mosaic Virus, a most impressive result obtained by fiber diffraction techniques.

The sixth day started with new protein structures, solved recently. Boris Vainshtein, Moscow, reported on a Mn-catalase and a human growth hormone; Ed Hough, Tromso, on the structure of phospholipase C, followed by Fred Vellieux, Groningen, on methylamine dehydrogenase, the first structure of an enzyme containing the recently discovered co-factor pirrolo quinoline quinone. Next, Uli Arndt, Cambridge, UK and Andy Howard, Genex, gave crystal clear descriptions of television area detector hardware and area detector software respectively. The afternoon saw an unbelievably impressive piece of work : the attempts to unravel the machinery of protein biosynthesis - the ribosome - at the atomic level. Thanks to careful purification techniques, shock-cooling and synchrotron radiation, data up to 5 A had been collected of a series of ribosome crystals some even with the growing polypeptide chain present inside the particle. New electron microscopy images of the ribosome lead to fascinating speculations regarding the position of the two tRNA's, the m-RNA and the emerging polypeptide chain. A 5A resolution X-ray structure would obviously be of tremendous interest but will take some time to obtain. Nevertheless, special heavy-atom derivatives had been synthetized with gold clusters as the central core. It will be fascinating to see how this work will progress (Ada Yonath and Klaus Bartels, Hamburg and Rehovot). The day was concluded by he description of single stranded DNA binding to DNA polymerase I (Lorena Beese, Yale) and a very nice summary of DNA-binding motifs in proteins plus insights into the catalytic mechanism of DNAse-I by Dietrich Suck, Heidelberg.

The next day saw descriptions of structures of potential medical importance: a beta-lactamase by Osnat Herzberg, Rockville; the binding of the sugar sialic acid to haemagglutinin, an outer membrane protein of influenza virus (Bill Weis, Harvard); the P2 myelin protein by Alwyn Jones, Uppsala. Joanne Rout gave a clear account of ice nucleation and proteins influencing this process while Martha Teeter first explained principles of single crystal neutron diffraction and then showed beautiful neutron diffraction results on the small protein crambin where also most of the water molecules positions had become localized.

The first session of the eighth day of the meeting was entirely tio the most abundant enzyme on this planet: 1,5-biphosphate carbocylase/oxygenase, in short "rubisco". The UCLA group described the structure determination and subunit arrangement in tobacco while the Uppsala group gave the first account of of the binding of a transition state analogue to the active site of the enzyme.

The "interactions" session saw the elucidation of the structure of a "two-headed" proteinase inhibitor complexed with chymotrypsin (Mark Grutter, Basel); the binding of iron and carbonate by transferring ( Richard Garratt, London) and the binding of a series of sugars and anions (such as sulphate) to the periplasmic binding proteins by Florante Quiocho, Houston. After the second poster session, exploration of protein structure and function by site directed mutagenesis was addressed by Phil Evans, Cambridge, UK describing the great insight obtained in catalysis and control of phosphofructokinase; Bauke Dijkstra, Groningen, showed the structure of a phospholipase A2 obtained by "loop transplantation" and having a 15-fold higher activity against micelles than native enzymes; Liang Tong, Berkeley, presented the first structure of the oncoprotein "p21" plus two of its mutants. The structure showed that virtually all predictions made were wrong - only some details were correctly predicted - but also that questions as to why mutations at position 12 are causing transformation of cells are still far from being completely understood.

The last day started with a description of attempts to alter properties of proteins by site directed mutagenesis. Tony Kossiakoff, Genentech, showed how newly engineered disulfides do not always have to be restricted to low energy forms. Even with high dihedral energy, new disulfides van stabilize a protein. Masarumi Matsumara, Eugene, showed many x-ray structures of mutants of T4 lysozyme at position 3. It was surprising to see how the creation of cavities was "solved" by filling it up by water molecules while the mutation Il3 -> Tyr caused the tyrosine side chain to swing out into solution. This will be tough to predict and model correctly ! In the next session Kiyoshi Nagai, Cambridge, UK, showed how amino acid substitution in the oxygen binding pocket affect structure and function of human haemoglobin.

In the Last Minute Results session fascinating results on the structure determination of SV40-virus, Foot-and-Mouth Disease Virus and protocatechuate hydroxylase were presented by Bob Liddington, Harvard, Dave Stuart, Oxford, and Doug Ohlendorg, Du Pont de Nemours, USA, respectively.

The protein structure, modelling and prediction session contained a report of Fred Cohen, S. Francisco on attempts of ab-initio protein structure prediction , starting from aminoacid sequence information and some experimentally obtained distance information. The problem appeared to be extremely difficult indeed. Tom Blundell, London, addressed a simpler problem : how to obtain an accurate model of a protein for which a sequence plus a number of related structures are known. An impressive set of programs have been developed which, together with a commercially available data base handler, allowed many protein modelling problems to be handled elegantly.

The final session of the meeting saw the structures of interleukin-1 and interleukin-2 be presented by John Priestle, Basel and Dave Mackay, Boulder, respectively. The last lecture was the magnificent structure determination of HLA-A2, a human class 1 histocompatibility antigen by Pamela Bjorkman, Harvard & Stanford. The lecture culminated in a model or part of a model T cell receptor interacting with the experimentally observed HLA structure with the "foreign" peptide, presented by the HLA to the receptor, sandwiched in between these two crucial molecules of the immune system.

This was the fitting finale of this successful meeting.

(from a document by Wim Hol, only slightly modified)

Extracted from several documents.
January 2008