Combined use of monochromatic and Laue diffraction techniques for macromolecular structure determination

A novel strategy of macromolecular structure analysis is described which combines the use of monochromatic scanning Laue (SCL) and white-beam Laue (WBL) diffraction techniques. It provides, when applied with an area detector with on-line capabilities, a means of interactively determining and optimizing experimental parameters; it further makes rapid data evaluation feasible, also with off-line detector systems. These new procedures have been applied to a protein structure, [beta]-trypsin, using a FAST area detector (Enraf-Nonius) and image plates (Fuji) on a double-focusing synchrotron beamline at DORIS. Structure factors, which were derived from FAST Laue data, were empirically scaled by comparing equivalent reflections in different wavelength bins. A 2Fo - Fc difference Fourier map, which was calculated at 1.8 Å resolution using these structure-factor moduli together with phases from the known structural model, showed well defined electron density distribution (R = 22%). Image-plate exposures showed diffraction to 1.2 Å resolution. The effect of crystal mosaicity on the maximum wavelength bandwidth for Laue exposures has been investigated. SCL techniques, which involve rapid scanning (with a crystal or multilayer monochromator or a tunable undulator) through a defined wavelength range, extend the applicability of Laue techniques to crystals with broadened mosaic spread

Fast high-performance liquid chromatographic purification of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

A procedure was established for the rapid isolation of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (PEPCK) from an overproducing strain. Overexpression was achieved by the transformation of yeast cells with the multicopy plasmid YEp352 harbouring the PEPCK structural gene. The enzyme was purified to homogeneity using first anion-exchange chromatography on Q-Sepharose followed by hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration on Sephacryl S200. The purified phosphoenolpyruvate carboxykinase was further characterized with respect to the molecular mass, displaying an apparent molecular mass corresponding to a tetrameric form

Accuracy in Laue X-ray diffraction analysis of protein structures

The accuracy in protein structure analysis based on Laue X-ray diffraction has been investigated for the example of two orthorhombic structures of bovine pancreatic trypsin (BPT). The precision in the Laue structure factors and the contrast in electron-density maps were used as criteria. A comparison with the results of previous analyses based on conventional crystal rotation methods showed that high resolution around 1.4 Å may be reached with both monochromatic and polychromatic techniques. Electron-density maps exhibited significantly lower contrast when calculated on the basis of Laue structure amplitudes, due to inefficient exploration of reciprocal space at low resolution by the Laue method even in the case of a broad bandwidth and inclusion of exposures from several different crystal orientations. Laue data were recorded on photographic film and processed using the program LAUEMAD [Bartunik & Borchert (1989). Acta Cryst. A45, 718-726]. The empirically derived wavelength scaling factors based on a comparison of equivalent reflection intensities were in good agreement with theoretical estimates over a broad wavelength range. One BPT structure was refined on the basis of Laue structure amplitudes (current R-factor 24% at 1.8 Å resolution)

Orthorhombic crystal form of bacteriorhodopsin nucleated on benzamidine diffracting to 3.6 A resolution.

Freshly formed benzamidine crystals were found to provide a suitable nucleation surface for crystallisation of bacteriorhodopsin. At 20 degrees C and 1% octylglucoside pseudohexagonal needles of bacteriorhodopsin nucleated on the benzamidine surface. At 4 degrees C and reduced detergent concentration (0.5%) a new, better-ordered orthorhombic crystal form of bacteriorhodopsin was formed by heterogeneous nucleation on benzamidine. Polarised absorption spectroscopy and flash photolysis experiments were used to show that the crystalline bacteriorhodopsin is photoactive in both forms. The M-intermediate absorbs maximally at 405nm and formation of M does not disturb the crystal lattice. The plate-shaped crystals diffract to a resolution of 3.6 A along the a and b directions and to 4.2 A in the c direction. The most likely space group of the crystals is C222 (a = 107.5 A, b = 117.0 A, c = 69.5 A). The crystals are built from layers of bacteriorhodopsin molecules that are tilted from the c axis by about 45 degrees. In addition, as a background to the discovery of the new crystal form, the influence of different detergents, additives and precipitants on the formation of pseudohexagonal needles is presented