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MAD Experiments (mad + experiment)
Selected AbstractsCrystallization and preliminary X-ray crystallographic analysis of the Sulfolobus solfataricus nucleotide-exchange factor 1,ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2005Paolo Arcari The nucleotide-exchange factor isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1,) consists of 90 residues and differs from eukaryal EF-1,s. The protein has been successfully crystallized using either microbatch-under-oil or vapour-diffusion methods. Crystals of native SsEF-1, diffract to 1.97,Å resolution and belong to space group P21212, with unit-cell parameters a = 106.46, b = 54.87, c = 44.03,Å. Diffraction data have also been collected from a selenomethionine derivative of SsEF-1, at 1.83,Å resolution. Model building using the phases derived from the MAD experiment is in progress. [source] Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experimentsACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2008Marc Schiltz The X-ray polarization anisotropy of anomalous scattering in crystals of brominated nucleic acids and selenated proteins is shown to have significant effects on the diffraction data collected at an absorption edge. For conventionally collected single- or multi-wavelength anomalous diffraction data, the main manifestation of the anisotropy of anomalous scattering is the breakage of the equivalence between symmetry-related reflections, inducing intensity differences between them that can be exploited to yield extra phase information in the structure-solution process. A new formalism for describing the anisotropy of anomalous scattering which allows these effects to be incorporated into the general scheme of experimental phasing methods using an extended Harker construction is introduced. This requires a paradigm shift in the data-processing strategy, since the usual separation of the data-merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking is explicitly modelled and refined and becomes a source of supplementary phase information. These ideas have been implemented in the phasing program SHARP. Refinements using actual data show that exploitation of the anisotropy of anomalous scattering can deliver substantial extra phasing power compared with conventional approaches using the same raw data. Examples are given that show improvements in the phases which are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment. It is argued that such gains, which come essentially for free, i.e. without the collection of new data, are highly significant, since radiation damage can frequently preclude the collection of a second-wavelength data set. Finally, further developments in synchrotron instrumentation and in the design of data-collection strategies that could help to maximize these gains are outlined. [source] Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with tetragonal hen egg-white lysozymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002Éric Girard A neutral gadolinium complex, Gd-HPDO3A, is shown to be a good candidate to use to obtain heavy-atom derivatives and solve macromolecular structures using anomalous dispersion. Tetragonal crystals of a gadolinium derivative of hen egg-white lysozyme were obtained by co-crystallization using different concentrations of the complex. Diffraction data from three derivative crystals (100, 50 and 10,mM) were collected to a resolution of 1.7,Å using Cu,K, radiation from a rotating anode. Two strong binding sites of the gadolinium complex to the protein were located from the gadolinium anomalous signal in both the 100 and 50,mM derivatives. A single site is occupied in the 10,mM derivative. Phasing using the anomalous signal at a single wavelength (SAD method) leads to an electron-density map of high quality. The structure of the 100,mM derivative has been refined. Two molecules of the gadolinium complex are close together. Both molecules are located close to tryptophan residues. Four chloride ions were found. The exceptional quality of the SAD electron-density map, only enhanced by solvent flattening, suggests that single-wavelength anomalous scattering with the Gd-HPDO3A complex may be sufficient to solve protein structures of high molecular weight by synchrotron-radiation experiments, if not by laboratory experiments. [source] Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007Andy Maraite Benzaldehyde lyase (BAL; EC 4.1.2.38) is a thiamine diphosphate (ThDP) dependent enzyme that catalyses the enantioselective carboligation of two molecules of benzaldehyde to form (R)-benzoin. BAL has hence aroused interest for its potential in the industrial synthesis of optically active benzoins and derivatives. The structure of BAL was previously solved to a resolution of 2.6,Å using MAD experiments on a selenomethionine derivative [Mosbacher et al. (2005), FEBS J.272, 6067,6076]. In this communication of parallel studies, BAL was crystallized in an alternative space group (P212121) and its structure refined to a resolution of 1.65,Å, allowing detailed observation of the water structure, active-site interactions with ThDP and also the electron density for the co-solvent 2-methyl-2,4-pentanediol (MPD) at hydrophobic patches of the enzyme surface. [source] Using X-ray absorption spectra to monitor specific radiation damage to anomalously scattering atoms in macromolecular crystallographyACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2007V. Oliéric Radiation damage in macromolecular crystals is not suppressed even at 90,K. This is particularly true for covalent bonds involving an anomalous scatterer (such as bromine) at the `peak wavelength'. It is shown that a series of absorption spectra recorded on a brominated RNA faithfully monitor the extent of cleavage. The continuous spectral changes during irradiation preserve an `isosbestic point', each spectrum being a linear combination of `zero' and `infinite' dose spectra. This easily yields a good estimate of the partial occupancy of bromine at any intermediate dose. The considerable effect on the near-edge features in the spectra of the crystal orientation versus the beam polarization has also been examined and found to be in good agreement with a previous study. Any significant influence of the (C,Br bond/beam polarization) angle on the cleavage kinetics of bromine was also searched for, but was not detected. These results will be useful for standard SAD/MAD experiments and for the emerging `radiation-damage-induced phasing' method exploiting both the anomalous signal of an anomalous scatterer and the `isomorphous' signal resulting from its cleavage. [source] | ||||||||||