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Phasing Methods (phasing + methods)
Selected AbstractsRetrieving low- and medium-resolution structural features of macromolecules directly from the diffraction intensities , a real-space approach to the X-ray phase problemACTA CRYSTALLOGRAPHICA SECTION A, Issue 6 2008Wu-Pei Su A simple mathematical algorithm is proposed to generate electron-density functions whose Fourier amplitudes match the diffraction intensities. The function is by construction everywhere positive. Using appropriate averaging procedures, the high-density regions of such functions could yield important structural information about macromolecular crystals. Trial calculations on protein crystals show that the protein envelope plus other structural motifs such as barrels and secondary structures could be recognized in the density maps. As such, the algorithm could provide a basis for new phasing methods or supplement existing phasing methods. [source] Features of the secondary structure of a protein molecule from powder diffraction dataACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Sebastian Basso Protein powder diffraction is shown to be suitable for obtaining de novo solutions to the phase problem at low resolution via phasing methods such as the isomorphous replacement method. Two heavy-atom derivatives (a gadolinium derivative and a holmium derivative) of the tetragonal form of hen egg-white lysozyme were crystallized at room temperature. Using synchrotron radiation, high-quality powder patterns were collected in which pH-induced anisotropic lattice-parameter changes were exploited in order to reduce the challenging and powder-specific problem of overlapping reflections. The phasing power of two heavy-atom derivatives in a multiple isomorphous replacement analysis enabled molecular structural information to be obtained up to approximately 5.3,Å resolution. At such a resolution, features of the secondary structure of the lysozyme molecule can be accurately located using programs dedicated to that effect. In addition, the quoted resolution is sufficient to determine the correct hand of the heavy-atom substructure which leads to an electron-density map representing the protein molecule of proper chirality. [source] Experimental phasing: best practice and pitfallsACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Airlie J. McCoy Developments in protein crystal structure determination by experimental phasing are reviewed, emphasizing the theoretical continuum between experimental phasing, density modification, model building and refinement. Traditional notions of the composition of the substructure and the best coefficients for map generation are discussed. Pitfalls such as determining the enantiomorph, identifying centrosymmetry (or pseudo-symmetry) in the substructure and crystal twinning are discussed in detail. An appendix introduces combined real,imaginary log-likelihood gradient map coefficients for SAD phasing and their use for substructure completion as implemented in the software Phaser. Supplementary material includes animated probabilistic Harker diagrams showing how maximum-likelihood-based phasing methods can be used to refine parameters in the case of SIR and MIR; it is hoped that these will be useful for those teaching best practice in experimental phasing methods. [source] Iodide-SAD, SIR and SIRAS phasing for structure solution of a nucleosome assembly proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009Manickam Yogavel The crystal structure of Plasmodium falciparum nucleosome assembly protein (PfNapL) was determined by iodide-SAD/SIRAS phasing methods using iodide-SAD data to 3.0,Å resolution and native data to 2.4,Å resolution. Halide-derivatized PfNapL crystals were obtained using the quick cryo-soaking method in which the native crystals were soaked in a cryosolution consisting of 500,mM NaI for a short period of 30,60,s and data were collected at an in-house X-ray source using Cu,K, radiation. Despite a low anomalous signal-to-noise ratio of <1.2 in the >3.5,Å resolution bin, the data were sufficient to determine the structure by SAD/SIR/SIRAS methods using the soaked iodides. Previously, structure solution had failed with both molecular-replacement and selenomethionine-derivatization techniques owing to reasons that are detailed in this work. The phasing at low resolution with three iodides per monomer with high temperature factors was successful using any of the SAD, SIR or SIRAS methods. [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] Crystallization of the hydantoin transporter Mhp1 from Microbacterium liquefaciensACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2008Tatsuro Shimamura The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P212121, unit-cell parameters a = 79.7, b = 101.1, c = 113.8,Å). A complete data set has been collected from a single crystal to a resolution of 2.85,Å with 64,741 independent observations (94% complete) and an Rmerge of 0.12. Further experimental phasing methods are under way. [source] | ||||||||||