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Near-atomic Resolution (near-atomic + resolution)
Selected AbstractsEM 3D contour maps provide protein assembly at the nanoscale within the neuronal porosome complexJOURNAL OF MICROSCOPY, Issue 1 2008W.J. CHO Summary The neuronal porosome complex, the secretory machinery at the plasma membrane of nerve terminals, is a 12,17-nm cup-shaped lipoprotein structure possessing a central plug. Since the porosome is a membrane associated, multi-protein complex measuring >650 kD, it has precluded generation of 3D crystals for x-ray diffraction studies, nor structural analysis at the atomic level using solution magnetic resonance spectroscopy. These limitations were partially overcome in the current studies, furthering our understanding of the porosome structure. Using atomic force microscopy, electron microscopy and electron density and 3D contour mapping, finally provides at the nanoscale, the structure and assembly of proteins within the neuronal porosome complex. Results from this study demonstrate a set of eight protein units lining the porosome cup, each connected via spoke-like elements to a central plug region within the structure. The isolation of intact porosomes for near-atomic resolution using cryo-electron diffraction measurements, is finally possible. [source] Near-atomic resolution structures of urate oxidase complexed with its substrate and analogues: the protonation state of the ligandACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010Laure Gabison Urate oxidase (uricase; EC 1.7.3.3; UOX) from Aspergillus flavus catalyzes the oxidation of uric acid in the presence of molecular oxygen to 5-hydroxyisourate in the degradation cascade of purines; intriguingly, catalysis proceeds using neither a metal ion (Fe, Cu etc.) nor a redox cofactor. UOX is a tetrameric enzyme with four active sites located at the interface of two subunits; its structure was refined at atomic resolution (1,Å) using new crystal data in the presence of xanthine and at near-atomic resolution (1.3,1.7,Å) in complexes with the natural substrate (urate) and two inhibitors: 8-nitroxanthine and 8-thiouric acid. Three new features of the structural and mechanistic behaviour of the enzyme were addressed. Firstly, the high resolution of the UOX,xanthine structure allowed the solution of an old structural problem at a contact zone within the tetramer; secondly, the protonation state of the substrate was determined from both a halochromic inhibitor complex (UOX,8-nitroxanthine) and from the H-atom distribution in the active site, using the structures of the UOX,xanthine and the UOX,uric acid complexes; and thirdly, it was possible to extend the general base system, characterized by the conserved catalytic triad Thr,Lys,His, to a large water network that is able to buffer and shuttle protons back and forth between the substrate and the peroxo hole along the reaction pathway. [source] UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructionsACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2009Xavier Siebert Electron microscopy of a macromolecular structure can lead to three-dimensional reconstructions with resolutions that are typically in the 30,10,Å range and sometimes even beyond 10,Å. Fitting atomic models of the individual components of the macromolecular structure (e.g. those obtained by X-ray crystallography or nuclear magnetic resonance) into an electron-microscopy map allows the interpretation of the latter at near-atomic resolution, providing insight into the interactions between the components. Graphical software is presented that was designed for the interactive fitting and refinement of atomic models into electron-microscopy reconstructions. Several characteristics enable it to be applied over a wide range of cases and resolutions. Firstly, calculations are performed in reciprocal space, which results in fast algorithms. This allows the entire reconstruction (or at least a sizeable portion of it) to be used by taking into account the symmetry of the reconstruction both in the calculations and in the graphical display. Secondly, atomic models can be placed graphically in the map while the correlation between the model-based electron density and the electron-microscopy reconstruction is computed and displayed in real time. The positions and orientations of the models are refined by a least-squares minimization. Thirdly, normal-mode calculations can be used to simulate conformational changes between the atomic model of an individual component and its corresponding density within a macromolecular complex determined by electron microscopy. These features are illustrated using three practical cases with different symmetries and resolutions. The software, together with examples and user instructions, is available free of charge at http://mem.ibs.fr/UROX/. [source] Crystallization and preliminary X-ray analysis of dihydrodipicolinate synthase from Clostridium botulinum in the presence of its substrate pyruvateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Sarah C. Atkinson In this paper, the crystallization and preliminary X-ray diffraction analysis to near-atomic resolution of DHDPS from Clostridium botulinum crystallized in the presence of its substrate pyruvate are presented. The enzyme crystallized in a number of forms using a variety of PEG precipitants, with the best crystal diffracting to 1.2,Å resolution and belonging to space group C2, in contrast to the unbound form, which had trigonal symmetry. The unit-cell parameters were a = 143.4, b = 54.8, c = 94.3,Å, , = 126.3°. The crystal volume per protein weight (VM) was 2.3,Å3,Da,1 (based on the presence of two monomers in the asymmetric unit), with an estimated solvent content of 46%. The high-resolution structure of the pyruvate-bound form of C. botulinum DHDPS will provide insight into the function and stability of this essential bacterial enzyme. [source] | ||||||||||