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- Macromolecular Crystallography
Macromolecular Crystallography
Introduction
Macromolecular crystallography (MX) at the ESRF has continued to thrive during 2002. Four beamlines (ID14-1, -2, -4; and ID29) have been available to the User Community with approximately 400 experimental sessions involving a total of around 1600 visitors to the beamlines being supported. Such levels of support could not have been achieved without the wholehearted commitment of the MX Group staff and their EMBL colleagues who together form the Joint Structural Biology Group, and the excellent backup of the ESRF User Office. Furthermore, ID13, the microfocus beamline, also contributes with a share of its beam time to macromolecular structure determination where an exceedingly small focus is required. In addition the Collaborating Research Group (CRG) beamlines continue to provide excellent support for their User Communities. The joint operation of BM14 by the Spanish and UK consortia has now been successfully concluded and the Spanish consortium will move to the newly rebuilt facilities on BM16 during 2003, with the UK consortium taking over sole responsibility for BM14. The French CRG beamline FIP (BM30) continues to enhance its performance with particular advances in the field of automation.
Technical enhancements of the MX Group beamlines continued apace with the major focus on improving the automation of both beamline alignment and data collection and also of crystal screening protocols. The policy of beamline upgrade and refurbishment has also continued with the rebuilding and commissioning of ID14-3 which will re-enter user operation in Spring 2003. The remodelling of ID14-3 is designed to produce the first fully-automatic MX beamline at the ESRF. Achieving this goal has necessitated the adoption of many new ideas for instrument control and X-ray beam diagnostics as well as the development of sophisticated and reliable software and hardware. S. Arzt and J. McCarthy in their article describe the scope and intentions of efforts currently underway in this area and it is expected that many of the developments arising from these will be deployed on the other MX Group beamlines starting in 2003. Construction of the ID23 beamline complex has also been started with the lead hutches erected and control cabins installed. ID23 was designed for construction in two stages. The first, the experimental facility, due to be available at the end of 2003, will consist of a MAD beamline with tuneability between 5 and 25 keV. We envisage that this beamline will also benefit from efforts elsewhere in the ESRF. Moreover, like ID14-3, it will provide a highly-automated environment.
Apart from providing a further platform for MX at the ESRF, the ID23 beamline also forms a crucial part of the Partnership for Structural Biology (PSB) collaboration. This joint enterprise will greatly augment and extend the structural biology programme of the ESRF, introducing better access to the expertise in molecular biology of the EMBL and IBS, and the neutron scattering capabilities of the ILL. Together, the four laboratories making up the PSB provide a resource, unique in Europe, for structural biology and genomics research, where industry will also play a major role. In their keynote addresses at the opening ceremony of the PSB, Prof. Fotis Kafatos, Director General of the EMBL, Prof. R. Douce, Director of IBS, and Prof. D. Stuart of Oxford University stressed the importance of the PSB to European structural biology research. A fuller account of the organisation and aims of the PSB is given in the General Introduction to these Highlights.
The Block Allocation Group (BAG) system has now been in operation for several years and has brought about a marked increase in the effectiveness with which ESRF beam-time is utilised by the MX User Community. The standard of publications arising from the use of the ESRF for MX remains extremely high. In this chapter, the editors have undertaken the task of reflecting this excellence by choosing to present highlight contributions that reflect the broad range of science carried out at the beamlines.
The outer membrane mitochondrial monoamine oxidase B (MOA B) plays a key role in the breakdown of neurotransmitters, is implicated in many neurological disorders and has long been a target for drug therapy. The three-dimensional structure of MAO B as determined by Mattevi et al. reveals an apparently novel method of membrane insertion for protein molecules and also provides the basis for future studies with the aim of designing novel drugs.
Membrane-bound proteins play an increasingly large part in the structural biology studied at the ESRF. Here they are also represented by the work of Hunte and Lange who report on the crystal structure of the electron transfer complex of Cytochrome c and the Cytochrome bc1 complex from yeast. Since the structure was obtained at physiological ionic strength there is evidence that the complex represents the true native electron transfer complex and therefore sheds significant light on the mechanism and regulation of the electron transfer process.
Blood clotting is essential to the maintenance of healthy blood circulation system. Gros et al. used data from ESRF beamlines to determine the structure of the complex between glycoprotein Ib and the blood protein von Willebrand factor (VWF) as amino acid mutations in either of the two moieties involved in the complex can cause bleeding disorders. The crystal structure of the complex described here allows both for the construction of a model for the mechanism of the adhesion of blood platelets at sites of bleeding (one of the first steps in clotting) as well as providing a framework for the structure-based design of novel therapeutical agents designed to treat diseases such as thrombosis.
Phosphate is a vital component of many cellular processes. In E. coli the uptake of phosphate is regulated by the signal transduction system based around PhoR and PhoB. M. Coll et al. have determined the interactions present at a key point of the signal transduction specific pathway namely the binding of PhoB to its specific DNA target.
R Fourme et al., using the high-energy physics beamline ID30, have undertaken studies on the effect of high pressures on protein and virus samples and structures. As well as a clear proof of principle the experiments described in this article offer tantalising glimpses of the possible exploitation of this technique to explore protein sub-states, to probe protein-protein interactions as a function of pressure and even, in favourable cases, to improve the quality of the diffraction patterns obtainable from crystals of macromolecules.
In-house research has also continued to develop in the last year. The work carried out by ESRF staff and their collaborators is represented here in three contributions.
D. Bourgeois, E. Fioravanti et al., using the technique of kinetic crystallography, have investigated the enzymatic reaction of the M. tuberculosis thymidylate kinase. They demonstrate the essential role played by a Mg2+ ion in catalysis and propose that potential anti-tuberculosis agents could be designed with this in mind. A. Imberty, E. Mitchell et al. have determined the structure of fucose binding lectin PA-IIL from P. aeruginosa, which is one of the major causes of mortality in cystic fibrosis sufferers. It is hoped that the structural information obtained from their analysis of the PA-IIL-fucose complex will provide a basis for design of efficient anti-bacterial compounds. Finally E. Hough et al. have determined the structure of the bovine lysosomal -mannosidase defects in which cause the lysosomal storage disorder -mannosidosis. Using a range of biophysical techniques including the determination of the three-dimensional structure of the enzyme using data collected at the ESRF these authors have been able to provide many insights into the molecular basis of the bovine and human forms of this disease.
Finally 2002 saw the departure of Peter Lindley as Director of Research. The MX group would like to thank him for the support and encouragement received during his tenure and wish him good luck in his "retirement" - don't work too hard!