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EMBL Hamburg SAXS studies of biological macromolecules in solution Previous and current research Fundamental biological processes, such as cell-cycle control, signalling, DNA duplication, gene expression and regulation and some metabolic pathways, depend on supra-molecular assemblies and their changes over time. There are objective difficulties in studying such complex systems, especially their dynamic changes, with high resolution structural techniques like X-ray crystallography or NMR. Small-angle X-ray scattering (SAXS) allows us to study native biological macromolecules from individual proteins to large complexes in solution, under nearly physiological conditions. SAXS not only provides low resolution 3D models of particle shapes but yields answers to important functional questions. In particular, it elucidates structural changes in response to variations in external conditions, protein-protein and protein-ligand interactions, and time-resolved studies permit one to characterise structural kinetics of assembly/dissociation or folding/unfolding. Our group runs beamline X33, dedicated to biological solution SAXS, at DESY’s storage ring, DORIS-III. We develop novel methods to construct 3D models of individual macromolecules and their complexes from the X-ray and neutron scattering data with advanced mathematical and bioinformatical approaches. These methods are extensively used to interpret the experimental data in collaborative user projects at X33. The rapidly-growing demand for SAXS in the biological community has led to a dramatic increase in the user turnover at X33 (over five times more than in 2000). The beamline was completely refurbished in 2004-2008, including automation of the experiment and data analysis procedures. The novel analysis methods and improved experimental facilities allow us to solve exciting biological problems in collaboration with the users of X33. Here, SAXS is used to study quaternary and domain structure of individual proteins, nucleic acids and their complexes, oligomeric mixtures, conformational transitions upon ligand binding, flexible systems and intrinsically unfolded proteins, processes of amyloid fibrillation and many other objects of high biological and medical importance (see figure for a recent example). Future projects and goals The present and future work of the group includes: • participation in numerous collaborative projects at X33 beamline employing SAXS to study the structure of a wide range of biological systems in solution; • development of novel methods and approaches for the reconstruction of tertiary and quaternary structure of macromolecules and their complexes from X-ray and neutron scattering data; • joint use of SAXS with other structural, biophysical and computational methods including neutron scattering, crystallography, NMR, electron microscopy, FRET, bioinformatics, etc; • maintenance and upgrade of the X33 beamline including automation of SAXS experiments and their analysis; • collaboration with the PETRA III group at EMBL Hamburg (opposite) in designing a new high-brilliance biological SAXS beamline at the third-generation PETRA storage ring at DESY. Dmitri Svergun PhD 1982, Institute of Crystallography, Moscow. Dr. of Science 1997, Institute of Crystallography, Moscow. At EMBL since 1991. Group leader since 2003. Structural organisation of Flavorubredoxin tetramer reconstructed from the SAXS patterns (intensity versus scattering angle; dots: experimental data, solid lines: computed from the models). Solution scattering study showed that: (i) the tetrameric flavodiiron (FDP) core (depicted in blue and yellow/orange) is more anisometric than the crystallographic models of the homologous FDPs; (ii) Rubredoxin (Rd) domains (magenta) are located on the periphery, being loosely connected to the core by extended linkers (green) and are freely available to participate in redox reactions with protein partners. Selected references Petoukhov, M. V., Vicente, J. B., Crowley, P. B., Carrondo, M. A., Teixeira, M., and Svergun, D. I. (2008). Quaternary structure of flavorubredoxin as revealed by synchrotron radiation small-angle X- ray scattering. Structure, 16, 128-136 Shukla, A., Mylonas, E., Di Cola, E., Finet, S., Timmins, P., Narayanan, T. & Svergun, D.I. (2008). Absence of equilibrium cluster phase in concentrated lysozyme solutions. Proc. Natl Acad. Sci. USA, 105, 5075-5080 She, M., Decker, C.J., Svergun, D.I., Round, A., Chen, N., Muhlrad, D., Parker, R. & Song, H. (2008). Structural basis of dcp2 recognition and activation by dcp1. Mol. Cell, 29, 337-39 Xu, X. F., Reinle, W. G., Hannemann, F., Konarev, P. V., Svergun, D. I., Bernhardt, R. & Ubbink, M. (2008). Dynamics in a pure encounter complex of two proteins studied by solution scattering and paramagnetic NMR spectroscopy. J. Am. Chem. Soc, 130, 6395-603 105
EMBL Research at a Glance 2009 Paul Tucker PhD 1972, University of Essex, Colchester. Postdoctoral research at the universities of Groningen, Leicester, the Australian National University in Canberra, and Exeter. Lecturer in Inorganic Chemistry, University of the West Indies. At EMBL Heidelberg 1985- 1998; at EMBL Hamburg since 1998. Structural studies of proteins from pathogens Previous and current research A major focus has been trying to understand, at a structural level, the mechanisms by which bacteria respond to external conditions. We are therefore interested in classical two-component systems and the way they regulate gene expression by responding to the environment. Mycobacterium tuberculosis (MtB) continues to pose a global health threat and is our organism of choice because understanding the way in which the bacteria responds to externally generated stress is a possible way to the discovery of novel antibiotics. In the last years we have extended our work to investigate the structure of proteins involved in regulating the production of the stress response sigma factor, σ F . The most interesting results have been on a protein that contains four regulatory domains, a sensor PAS domain, a kinase domain (the anti-sigma factor), a phosphatase domain and an antisigma factor antagonist domain. In this system we are beginning to understand the structural basis of the regulation mechanism, and, from the structural work, have some idea as to what signal is used to switch on σ F dependent genes. Two structures are shown in figure 1. A second area of study has been on proteins that are involved in replication of the genome of a variety of RNA viruses. Our goal is still to obtain structural information on the RNA dependent RNA polymerase domains of the caliciviridae, the flaviviridae and, more importantly, a vesiculovirus. We have also continued our work on the structural and functional aspects of single-stranded DNA binding proteins found in the dsDNA viruses, and are interested in how these proteins interact with other components of the replication machinery (figure 2). A third focus, largely with Staff Scientist Santosh Panjikar and the Weiss team (opposite), remains on improving the structure determination process by, for example, developing improved phasing methods and automating structure determination. Santosh Panjikar is also actively engaged in elucidated the enzymes of the biosynthetic pathway of various indole alkaloids in Indian medicinal plants. Future projects and goals We will continue our work on the unusual actinobacterial two-component system (PdtaR/PdtaS) we discovered a few years ago, by determining the structure of the sensor domain of the (cytosolic) histidine kinase. We will continue our structural work on domains of the coronavirus NSP3 protein, as part of a comparative analysis of these domains and their organisation in the different classes of the coronaviridae. Figure 1: The structures of the PAS (sensor) domain (blue), the kinase (red) and phosphatase (green) domains of the Mycobacterium tuberculosis regulatory protein Rv1364c. Figure 2: The solution structure (represented by grey spheres) from small-angle X-ray scattering of a dimer of the Epstein-Barr virus single stranded DNA binding protein BalF2. Superimposed is a homology model derived from the equivalent Herpes Simplex Virus 1 protein, ICP8. We are expecting to push forward our structural work on lipid binding proteins from nematodes. There is a complete lack of structural information on this class of proteins and, for parasitic nematodes, they are essential for the viability of the organism in the host. Selected references King-Scott, J., Nowak, E., Mylonas, E., Panjikar, S., Roessle, M., Svergun, D.I. & Tucker, P.A. (2007). The structure of a full-length response regulator from Mycobacterium tuberculosis in a stabilised 3D domain-swapped, activated state. J. Biol. Chem., 282, 37717-29 Stockigt, J. & Panjikar, S. (2007). Structural biology in plant natural product biosynthesis – architecture of enzymes from monoterpenoid indole and tropane alkaloid biosynthesis. Natural Product Reports, 2, 1382-100 Tucker, P.A., Jens, E.N. & Morth, P. (2007). Two-component systems of M. tuberculosis: structure-based approaches. Methods Enzymol., 23, 79-501 Tucker, P.A. & Sallai, L. (2007). The AAA+ superfamily-a myriad of motions. Curr. Opin. Struct. Biol., 17, 61-652 106
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