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EMBL Hamburg Macromolecular crystallography Accurate modelling of biological processes depends on an intimate knowledge of the macromolecules involved. The availability of models of the interacting components allows a deep understanding of the basic processes that underlie human disease and all other cellular events. One recent example is a basis for neurodegenerative pathologies provided by the structure of a stable serpin dimer (Yamasaki et al., 2008, Nature). Macromolecular structures are produced by a large number of independent scientists, all of whom approach specific problems in their own manner. Methods are needed to establish objective structure determination and quality assessment, which will impact further system-wide analysis. The group’s major activity is development of novel approaches and software tools for high-throughput 3D structure determination and detailed interpretation of biological macromolecules and their complexes. Specific attention is given to extension of the current limits of applicability of MX and its use with complementary techniques. Previous and current research In MX, availability of comprehensive software packages has a major impact on structural biology. Traditionally crystallographic model building is done by expert users with the aid of specialised graphics software. The automation of this process, first exemplified in the ARP/wARP package (Langer et al., 2008, Nature Protocols), was promptly followed by developments worldwide. ARP/wARP has been used extensively for thousands of structure determinations and is often used as a benchmark to evaluate the quality of electron density obtained by new methods; it has been integrated into many crystallographic software pipelines as the default model-building engine. A wide spectrum of ARP/wARP functionalities (e.g. Hattne et al., 2008, Acta Cryst.) makes the software particularly attractive to users. We continue the developments of the automated structure determination pipeline AutoRickshaw (led by S. Panjikar in collaboration with the Tucker group and Weiss team (pages 106 and 107)), which uses an artificial intelligence decision-making system (Manjasetty et al., 2008, Proteomics). With the help of ARP/wARP and other software, AutoRickshaw produces an interpretable electron density map and a partial structure shortly after data acquisition. At our MX beamlines the users receive immediate feedback whether the measured data are of sufficient quality for successful structure determination. Our group has initiated the installation of the EMBL Hamburg advanced computational facilities, where the in-house, beamline and external users can carry out 3D structure determination remotely. These services for AutoRickshaw and particularly ARP/wARP have become increasingly popular (see figure). At the other end of the scale, we continue pushing the limits of model interpretation at ultra-high resolution. Using quantum chemical calculations we were able to decipher subtle changes in the active site residues upon substrate binding in Hydroxynitrile lyase (Schmidt et al., 2008, J. Biol. Chem.). We showed that His235 of the catalytic triad must be protonated in order for catalysis to proceed. We also detected considerable pKa shifts that had been hypothesised earlier. Future projects and goals Victor S. Lamzin PhD 1987, Moscow State University. Scientist, Inst. Biochemistry, Russian Academy of Sciences, Moscow, until 1991. Postdoctoral research at EMBL Hamburg until 1995; Staff Scientist until 1997. Group leader and Deputy Head since 1997. User numbers of the ARP/wARP and AutoRickshaw remote computation facilities are increasing rapidly. In 2008 the facilities were used by more than 600 people, and over 1,000 macromolecular structures were solved. In the future we will witness the further integration of MX with complementary techniques, and the underlying methodology will face a number of challenges. Crystalline samples are already obtained from increasingly complex biological systems and are becoming smaller and more difficult to handle, with data harder to extract. The group’s activities will cast within the anticipated trend and aim at furthering the understanding of molecular biological processes by increasing the amount of 3D information that can be obtained from biological macromolecules. Selected references Yamasaki, M., Li, W., Johnson, D.J. & Huntington, J.A. (2008). Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization. Nature, 55, 1255-8 Hattne, J. & Lamzin, V.S. (2008). Pattern-recognition-based detection of planar objects in three-dimensional electron-density maps. Acta Crystallogr. D Biol. Crystallogr., 6, 83-82 Langer, G., Cohen, S.X., Lamzin, V.S. & Perrakis, A. (2008). Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc., 3, 1171-1179 Manjasetty, B.A., Turnbull, A.P., Panjikar, S., Bussow, K. & Chance, M.R. (2008). Automated technologies and novel techniques to accelerate protein crystallography for structural genomics. Proteomics, 8, 612-625 Schmidt, A., Gruber, K., Kratky, C. & Lamzin, V.S. (2008). Atomic resolution crystal structures and quantum chemistry meet to reveal subtleties of hydroxynitrile lyase catalysis. J. Biol. Chem., 283, 21827-21836 101
EMBL Research at a Glance 2009 Wolfram Meyer- Klaucke PhD 1996, Hamburg University. Postdoctoral research at the Medical University of Lübeck. At EMBL Hamburg since 1998. Team leader since 2000. Trace elements in biological systems Previous and current research novel antibiotics. Recently, we solved the crystal structure of FurB from Mycobacterium tuberculosis, which together with biochemical and spectroscopic data allowed us to propose the functional role of this protein. Although the overall fold of FurB with an N-terminal DNA binding domain and a C-terminal dimerisation domain is conserved among the Zur/Fur family, large differences in the spatial arrangement of the two domains with respect to each other can be observed. The biochemical and spectroscopic analysis revealed that M. tuberculosis FurB is Zn(II)- dependent and is likely to control genes involved in the bacterial zinc uptake. The combination of the structural, spectroscopic and biochemical results enabled us to determine the structural basis for functional differences in this important family of bacterial regulators (Lucarelli, 2007). Trace elements such as metals play a key role in the structure and function of about 30% of all proteins. Many biocatalytic processes depend on the presence of metal ions. Our research deals with metal functionality, binding and selectivity in biological systems. The group’s projects combine structural techniques with molecular biology, biochemistry and further methods aiming at a complete understanding of metal related biological processes. Apart from methods development (Korbas, 2006; Wellenreuther, 2007), current research includes: Proteins of the metallo-β-lactamase superfamily. This superfamily, with an active site capable of binding up to two metal ions, catalyses a variety of enzymatic processes. Beside the global metal binding motif the overall fold of α-sheets and β-helices is conserved within the superfamily (Kostelecky 2006). Their physiological importance varies from putative association with cancer and antibiotic resistance to different roles in cellular detoxification. Metal regulation. Members of the ferric/zinc uptake regulator (Fur/Zur) family are the central metal-dependent regulator proteins in many Gram-negative and -positive bacteria. They are responsible for the control of a wide variety of basic physiological processes and the expression of important virulence factors in human pathogens. Therefore, Fur has gathered significant interest as a potential target for New metal binding motifs. Hydrogenases are enzymes that catalyse the reversible oxidation of molecular hydrogen. Their structure and catalytic mechanism are of considerable applied interest as models for the development of efficient catalysts for hydrogen-fueled processes. Despite intensive efforts, however, the understanding of how hydrogenases react with H2 is only in its infancy. The only mononuclear hydrogenase, Hmd, harbours an iron containing cofactor of yet unknown structure. X-ray absorption spectroscopy determined two CO, one sulphur, and two nitrogen/oxygen atoms coordinated to the iron, the sulphur ligand being most probably provided by the protein. In active Hmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction (Korbas, 2006). Future projects and goals Structural models for the iron site in mononuclear hydrogenase under different biochemical conditions. In addition to the metal specificity of proteins we will focus on the regulation of metal concentrations in cells. At present we combine structural analysis (e.g. XAS, SAXS, protein crystallography) and biochemical methods (element analysis, isothermal calorimetry, enzyme kinetics), but spatial resolved spectroscopy and advanced spectroscopic techniques will play an increasing role (Wellenreuther et al., 2009). Selected references Wellenreuther, G., Cianci, M., Tucoulou, R., Meyer-Klaucke, W. & Haase, H. (2009). The ligand environment of zinc stored in vesicles. Biochem. Biophys. Res. Commun., 380, 198-203 Küpper, F.C., Carpenter, L.J., McFiggans, G.B., Palmer, C.J., Waite, T.J., Boneberg, E.M. et al. (2008). Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry. Proc. Natl Acad. Sci. USA, 105, 695-8 Shima, S., Pilak, O., Vogt, S., Schick, M., Stagni, M.S., Meyer- Klaucke, W., Warkentin, E., Thauer, R.K. & Ermler, U. (2008). The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site. Science, 321, 572-575 Lucarelli, D., Russo, S., Garman, E., Milano, A., Meyer-Klaucke, W. & Pohl, E. (2007). Crystal structure and function of the zinc uptake regulator FurB from Mycobacterium tuberculosis. J. Biol. Chem., 282, 991-9922 102
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