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Anomalous Scatterers (anomalous + scatterer)
Selected AbstractsUsing barium ions for heavy-atom derivatization and phasing of xylanase II from Trichoderma longibrachiatumACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2007Natalia Moiseeva This paper describes the use of barium chloride to produce a heavy-atom derivative of xylanase II crystals from Trichoderma longibrachiatum, which was obtained either by cocrystallization or soaking. SAD phasing led to interpretable electron-density maps that allowed unambiguous chain tracing. In the best case, with a data set collected at 9.5,keV, 88% of the residues were built, with 83% of the side chains assigned. The barium ions are found to mainly interact with main-chain carbonyl groups and water molecules. It is suggested that barium ions could also be used as a potential anomalous scatterer in the quick cryosoaking procedure for phasing. [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] The revenge of the Patterson methods.JOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2 2007The Patterson techniques, recently developed by the same authors for the ab initio crystal structure solution of proteins, have been applied to single and multiple anomalous diffraction (SAD and MAD) data to find the substructure of the anomalous scatterers. An automatic procedure has been applied to a large set of test structures, some of which were originally solved with remarkable difficulty. In all cases, the procedure automatically leads to interpretable electron density maps. Patterson techniques have been compared with direct methods; the former seem to be more efficient than the latter, so confirming the results obtained for ab initio phasing, and disproving the common belief that they could only be applied to determine large equal-atom substructures with difficulty. [source] Evaluation of SnB for the location of anomalous scattering atoms in SAD/MAD phasingJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 6 2003Jun Wang Sixteen existing single-wavelength/multi-wavelength anomalous diffraction (SAD/MAD) data sets with a broad range of crystallographic properties have been used to investigate the various parameters in SnB with the goal of finding the optimum values for locating the positions of anomalous scatterers. The results of the analysis indicate some changes in default parameters that may be useful for non-routine and difficult cases. [source] Clear strategy screens for macromolecular crystallizationJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2 2001Andrzej Marek Brzozowski The development of high-throughput crystallography combined with the wealth of already accumulated information about protein crystallization properties requires constant revision of current crystallization screening procedures. Two complementary 6 × 4 matrix `clear strategy screens' (CSS) have been developed and tested on a number of previously non-crystallized proteins. The screens yielded diffraction-quality crystals of a wide range of proteins (enzymes, transcription factors, structural proteins, etc.) in cases where the applications of commercially available screens were unsuccessful. Both their inherently simple design and their flexible nature provide an experimenter with a logical platform for further modification and optimization. Furthermore, the screens facilitate cryoprotection and potential incorporation of anomalous scatterers for multiple/single-wavelength anomalous dispersion (MAD/SAD) experiments. [source] Use of Cr K, radiation to enhance the signal from anomalous scatterers including sulfurJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3-2 2000Witek Kwiatkowski The anomalous signals from scatterers such as sulfur (S) and arsenic (As) were compared in diffraction data sets collected from an X-ray source with three different targets, Au, Cu and Cr, on a multi-target rotating anode. HIV-1 integrase crystals served as the test case for this study. The crystalline specimen of HIV-1 integrase contains in each protein molecule two As atoms, each covalently bound to a cysteine S atom, and two additional S atoms derived from methionine. It was found that the Cr K, radiation gave the clearest peaks in anomalous difference Fourier maps, although the signal-to-noise ratios of the anomalous signal for the Cu K, and Cr K, data were similar but better than that for Au L,. This result was in spite of the fourfold higher flux from the Cu anode versus the Cr anode. For all three X-ray wavelengths, anomalous difference Fourier maps calculated with bias-removed phases derived from the known atomic model revealed clear peaks at the two As sites. However, only in the map calculated using the Cr K, data were both peaks of the expected ellipsoidal shape, enveloping the As atom and the adjacent S atom. None of the S sites was apparent in difference maps calculated using the Au L, data. The ability to enhance the S-derived anomalous signal using Cr K, radiation has particularly useful applications in the structure determination of proteins, for example in resolving ambiguities in the chain tracing of a protein with numerous disulfide bonds and in assigning amino acid identities. Additionally, anomalous difference Patterson maps calculated from the Cr K, data were sufficiently clear to identify the As-related peaks. These results form the groundwork for in-house phase determination with the multi-wavelength anomalous diffraction method. [source] Enhancing MAD FA data for substructure determinationACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Hongliang Xu Heavy-atom substructure determination is a critical step in phasing an unknown macromolecular structure. Dual-space (Shake-and-Bake) recycling is a very effective procedure for locating the substructure (heavy) atoms using FA data estimated from multiple-wavelength anomalous diffraction. However, the estimated FA are susceptible to the accumulation of errors in the individual intensity measurements at several wavelengths and from inaccurate estimation of the anomalous atomic scattering corrections f, and f,,. In this paper, a new statistical and computational procedure which merges multiple FA estimates into an averaged data set is used to further improve the quality of the estimated anomalous amplitudes. The results of 18 Se-atom substructure determinations provide convincing evidence in favor of using such a procedure to locate anomalous scatterers. [source] Away from the edge II: in-house Se-SAS phasing with chromium radiationACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2005Hao Xu Recently, the demands of high-throughput macromolecular crystallography have driven continuous improvements in phasing methods, data-collection protocols and many other technologies. Single-wavelength anomalous scattering (SAS) phasing with chromium X-ray radiation opens a new possibility for phasing a protein with data collected in-house and has led to several successful examples of de novo structure solution using only weak anomalous scatterers such as sulfur. To further reduce data-collection time and make SAS phasing more robust, it is natural to combine selenomethionine-derivatized protein (SeMet protein) with Cr,K, radiation to take advantage of the larger anomalous scattering signal from selenium ( = 2.28 e,) compared with sulfur ( = 1.14 e,). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr,K, radiation. Each protein molecule contains eight selenomethionine residues in 148 amino-acid residues, providing a calculated Bijvoet ratio of about 3.5% at the Cr,K, wavelength. A single data set to 2.2,Å resolution with approximately ninefold redundancy was collected using an imaging-plate detector coupled with a Cr source. Structure solution, refinement and deposition to the Protein Data Bank were performed within 9,h of the availability of the scaled diffraction data. The procedure used here is applicable to many other proteins and promises to become a routine pathway for in-house high-throughput crystallography. [source] Substructure solution with SHELXDACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-2 2002Thomas R. Schneider Iterative dual-space direct methods based on phase refinement in reciprocal space and peak picking in real space are able to locate relatively large numbers of anomalous scatterers efficiently from MAD or SAD data. Truncation of the data at a particular resolution, typically in the range 3.0,3.5,Å, can be critical to success. The efficiency can be improved by roughly an order of magnitude by Patterson-based seeding instead of starting from random phases or sites; Patterson superposition methods also provide useful validation. The program SHELXD implementing this approach is available as part of the SHELX package. [source] MAD phasing: probabilistic estimate of |Foa|ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002Maria Cristina Burla The method of the joint probability distribution function is applied in order to estimate the structure-factor moduli of the anomalous scatterer substructure. The two-wavelength case is examined: the prior knowledge of the moduli is used to predict the value of |Foa| arising from the normal scattering of the anomalous scatterers. The conclusive formula is applied to ideal and to real cases: evidence of the usefulness of the approach is obtained. [source] Novel approach to phasing proteins: derivatization by short cryo-soaking with halidesACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2000Zbigniew Dauter A quick (less than 1,min) soak of protein crystals in a cryo-solution containing bromide or iodide anions leads to incorporation of these anomalous scatterers into the ordered solvent region around the protein molecules. These halide anions provide a convenient way of phasing through their anomalous scattering signal: bromides using multiwavelength anomalous dispersion (MAD) and bromides and/or iodides using single-wavelength anomalous dispersion (SAD) or single isomorphous replacement with anomalous scattering (SIRAS) methods. This approach has been tested successfully on four different proteins and has been used to solve the structure of a new protein of molecular weight 30,kDa. [source] | ||||||||||