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MAD Phasing (mad + phasing)
Terms modified by MAD Phasing Selected AbstractsA case of structure determination using pseudosymmetryACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Sergei Radaev Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: am = bo,co, bm = ao and cm = ,2co,bo. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement. [source] MAD phasing using the (Ta6Br12)2+ cluster: a retrospective studyACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2008Oliwia Pasternak The crystal structure of cytokinin-specific binding protein (CSBP) containing four independent molecules with 4 × 155 = 620 residues in the asymmetric unit of the P64 unit cell has been solved by three-wavelength MAD using 1.8,Å resolution data recorded from a crystal derivatized with the dodecabromohexatantalum cation (Ta6Br12)2+. The diffraction data contained a very strong anomalous signal (allowing successful phasing even using peak SAD data alone) despite the fact that the five (Ta6Br12)2+ clusters found in the asymmetric unit have low occupancy (about 0.3). The derivative structure has been successfully refined to R = 0.158, providing interesting details on the geometry of the (Ta6Br12)2+ cluster, its interactions with the protein and on the backsoaking of a cytokinin ligand that was originally part of a CSBP,cytokinin complex in the native crystals used for (Ta6Br12)2+ derivatization. A simulation analysis of the phasing power of the (Ta6Br12)2+ ions at artificially imposed resolution limits shows that it is not possible to resolve the individual Ta atoms if the dmin limit of the data is higher than 2.9,Å. Additionally, for successful Ta identification the (Ta6Br12)2+ complex should be specifically bound and ordered. Good binding at the protein surface is facilitated by the presence of acidic groups, indicating higher pH buffer conditions to be preferable. In addition, the water channels in the crystal should be sufficiently wide (at least 11,Å) to allow free diffusion of the (Ta6Br12)2+ ions on soaking. A retrospective look at the initial molecular-replacement calculations provides interesting insights into how the peculiar packing mode and strong bias of the molecular-replacement-phased electron-density maps had hindered successful solution of the structure by this method. [source] Escherichia coli MltA: MAD phasing and refinement of a tetartohedrally twinned protein crystal structureACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2005Thomas R. M. Barends Crystals were grown of a mutant form of the bacterial cell-wall maintenance protein MltA that diffracted to 2.15,Å resolution. When phasing with molecular replacement using the native structure failed, selenium MAD was used to obtain initial phases. However, after MAD phasing the crystals were found to be tetartohedrally twinned, hampering correct space-group determination and refinement. A refinement protocol was designed to take tetartohedral twinning into account and was successfully applied to refine the structure. The refinement protocol is described and the reasons for the failure of molecular replacement and the success of MAD are discussed in terms of the effects of the tetartohedral twinning. [source] MAD phasing: choosing the most informative wavelength combinationACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004Maria Cristina Burla Two algorithms are described for limiting data resolution and for predicting the most informative wavelength combinations in MAD techniques. Both have been successfully tested using experimental data from a large set of test structures. [source] Crystallization and phasing of focal adhesion protein 52 from Gallus gallusACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Imre Tör Focal adhesion protein 52 (FAP52) is a multidomain adaptor protein of 448 amino acids characterized as an abundant component of focal adhesions. FAP52 binds to filamin via its N-terminal ,-helical domain, suggesting a role in linking focal adhesions to the actin-based cytoskeleton. The recombinant protein was crystallized using the hanging-drop vapour-diffusion method, which yielded two crystal forms. Native data were collected from both crystal forms to 2.8 and 2.1,Å resolution, respectively. For one of the crystal forms, initial MAD phasing was successfully performed using two data sets from xenon-derivatized crystals. The derivative data sets were collected using softer X-rays of 1.5 and 1.9,Å wavelength. Preliminary structural analysis reveals the presence of a dimer in the asymmetric unit. [source] Structure of a conserved CoA-binding protein synthesized by a cell-free systemACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Takashi Wada TT1466 is a hypothetical protein from the extremely thermophilic bacterium Thermus thermophilus HB8 and is highly conserved in bacteria and archaea. The selenomethionyl protein was synthesized by a cell-free system and the crystal structure was determined at 2.0,Å by MAD phasing. A native crystal was used for structure refinement to 1.7,Å. The structure is highly homologous to that of the CoA-binding domain of the succinyl-CoA synthetase from Escherichia coli, despite the protein having only 14% sequence identity to this domain. An isothermal titration calorimetry experiment was performed to investigate whether TT1466 binds CoA and revealed high-affinity CoA binding of TT1466. [source] X-ray-induced debromination of nucleic acids at the Br,K absorption edge and implications for MAD phasingACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2002E. Ennifar Multi-wavelength anomalous dispersion (MAD) using brominated derivatives is considered a common and convenient technique for solving chemically synthesized nucleic acid structures. Here, it is shown that a relatively moderate X-ray dose (of the order of 5 × 1015,photons,mm,2) can induce sufficient debromination to prevent structure determination. The decrease in bromine occupancy with radiation dose can be accounted for by a simple exponential, with an estimated rate constant at the absorption-peak wavelength, 7.4,(0.8),MGy, that is not significantly different from its value at the absorption-edge wavelength, 9.2,(2.6),MGy (the given e.s.d.s assess the relative closeness of the two values, not their absolute accuracy, which is probably worse). Chemically, these results (and others) are consistent with bromine cleavage resulting from direct photodissociation and/or from the action of free electrons, rather than from the action of hydroxyl radicals originating from water dissociation. The free bromine species (Br,) diffuse too quickly, even in amorphous ice around 100,K, to allow the determination of a diffusion coefficient. From a practical point of view, it is suggested that a single data collection with a crystal consisting of iodinated instead of brominated derivatives could provide both anomalous scattering and SIR phase information by the progressive cleavage of iodine. [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] 3-Carboxy- cis,cis -muconate lactonizing enzyme from Neurospora crassa: MAD phasing with 80 selenomethioninesACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2002Michael C. Merckel The structure of 3-carboxy- cis,cis -muconate lactonizing enzyme from Neurospora crassa was determined at 3.0,Å resolution. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using crystals of fully substituted selenomethionine protein. However, the structure determination was not routine owing to the relatively poor quality of the diffraction data and the large number of twofolds in the unit cell. Eventually, 80 selenium sites were identified by the combined use of direct methods and real-space map interpretation. This represents one of the largest selenium substructures solved and used for phasing. Some of the difficulties in the structure determination and the methods used to address them are discussed. [source] Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaIACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2001William T. Watson Acyl-homoserine- l -lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S -adenosyl- l -methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P43, with unit-cell parameters a = b = 66.40, c = 47.33,Å. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5,Å and the perrhenate ion binding sites and liganding residues have been identified. [source] Crystallization and initial crystallographic analysis of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosaACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000Catherine A. Regni The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) catalyzes the conversion of mannose 6-phosphate to mannose 1-phosphate in the second step of the alginate biosynthetic pathway of Pseudomonas aeruginosa. PMM/PGM has been crystallized by hanging-drop vapor diffusion in space group P212121. Crystals diffract to 1.75,Å resolution on a synchrotron X-ray source under cryo-cooling conditions. PMM/PGM substituted with selenomethionine has been purified and crystallizes isomorphously with the native enzyme. Structure determination by MAD phasing is under way. Because of its role in alginate biosynthesis, PMM/PGM is a potential target for therapeutic inhibitors to combat P. aeruginosa infections. [source] Crystallization and preliminary X-ray crystallographic analysis of MinE, the cell-division topological specificity factor from Helicobacter pyloriACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Gil Bu Kang Cell division in Gram-negative bacteria is driven by the formation of an FtsZ ring at the division site. MinE regulates the proper placement of the FtsZ ring at mid-cell by blocking the inhibitory action of the MinCD complex. Diffraction data were collected at 2.8,Å resolution from a native crystal of full-length Helicobacter pylori MinE. The crystal belonged to space group P64. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient was 2.58,Å3,Da,1, which corresponds to a solvent content of 52.3%. For MAD phasing, a four-wavelength data set was collected at 3.0,Å resolution. [source] Crystallographic characterization of a multidomain histidine protein kinase from an essential two-component regulatory systemACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Haiyan Zhao YycGF is a highly conserved two-component signal transduction system that is specific to low-G+C Gram-positive bacteria, including many important human pathogens. It has been recognized as a crucial regulatory system for cell-wall metabolism. YycG, the histidine protein kinase of this system, is a multidomain transmembrane protein. The truncated cytoplasmic portion of YycG from Bacillus subtilis encompassing the PAS domain, the dimerization domain and the catalytic domain was expressed, purified and crystallized. X-ray data were collected to 2.8,Å resolution with a completeness of 98.2% and an overall Rmerge of 5.6%. The crystals belonged to space group P61 or P65, with unit-cell parameters a = 135.0, c = 133.0,Å. The selenomethionine-substituted version of the protein was crystallized and X-ray data were collected to 3.6,Å resolution for subsequent MAD phasing. [source] Selenium Derivatization of Nucleic Acids for X-Ray Crystal-Structure and Function StudiesCHEMISTRY & BIODIVERSITY, Issue 4 2010Jia Sheng Abstract It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se-Met) strategy). This selenium derivatization strategy via MAD (multi-wavelength anomalous dispersion) phasing has revolutionized protein X-ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X-ray crystal-structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O-atom at the positions 2,, 4,, 5,, and in nucleobases and non-bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid-phase chemical synthesis and enzymatic methods, and the Se-derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein,nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2,-position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom-specific substitution of the nucleotide O-atom, better stability under X-ray radiation, and structure isomorphism. Therefore, our Se-derivatization strategy has great potentials to provide rational solutions for both phase determination and high-quality crystal growth in nucleic-acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site-specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se-derivatization strategy in nucleic acid and protein research are also described in this review. [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] A large-scale, high-efficiency and low-cost platform for structural genomics studiesACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2006Xiao-Dong Su A large-scale, high-efficiency and low-cost platform based on a Beckman Coulter Biomek FX and custom-made automation systems for structural genomics has been set up at Peking University, Beijing, People's Republic of China. This platform has the capacity to process up to 2000 genes per year for structural and functional analyses. Bacillus subtilis, a model organism for Gram-positive bacteria, and Streptococcus mutans, a major pathogen of dental caries, were selected as the main targets. To date, more than 470 B. subtilis and 1200 S.,mutans proteins and hundreds of proteins from other sources, including human liver proteins, have been selected as targets for this platform. The selected genes are mainly related to important metabolism pathways and/or have potential relevance for drug design. To date, 40 independent structures have been determined; of these 11 are in the category of novel structures by the criterion of having less than 30% sequence identity to known structures. More than 13 structures were determined by SAD/MAD phasing. The macromolecular crystallography beamline at the Beijing Synchrotron Radiation Facility and modern phasing programs have been crucial components of the operation of the platform. The idea and practice of the genomic approach have been successfully adopted in a moderately funded structural biology program and it is believed this adaptation will greatly improve the production of protein structures. The goal is to be able to solve a protein structure of moderate difficulty at a cost about US $10,000. [source] | ||||||||||