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SAD Phasing (sad + phasing)
Kinds of SAD Phasing Selected AbstractsStructure of d -tyrosyl-tRNATyr deacylase using home-source Cu,K, and moderate-quality iodide-SAD data: structural polymorphism and HEPES-bound enzyme statesACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Manickam Yogavel d -Tyrosyl-tRNATyr deacylase (DTD) is an editing enzyme that removes d -amino acids from mischarged tRNAs. The crystal structure of Plasmodium falciparum DTD (PfDTD) was determined using the iodide-SAD phasing method. Iodide-derivatized PfDTD crystals were obtained using the quick cryo-soaking procedure in which native crystals were soaked for a short period of 10,30,s in cryoprotectant solution containing 0.2,1,M NaI. Iodide-SAD data sets were collected to 3.3 and 2.74,Å resolution from PfDTD crystals that belonged to two different space groups, P43 and P1, using an in-house X-ray copper-anode source. This is the first report to detail structure solution using low iodide anomalous signal, modest resolution and redundancy and average solvent content for SAD phasing of 984 and 1312 amino acids in the triclinic P1 and tetragonal P43 space groups, respectively. A total of 85% and 56% of the residues were automatically built into the iodide-phased electron-density maps using PHENIX AutoBuild. The structure of HEPES-bound PfDTD was subsequently determined by molecular replacement and refined to 2.83,Å resolution. The crystals obtained from various batches of crystallization trials of PfDTD exhibited polymorphism in terms of belonging to different crystal forms and space groups. Even within a given crystal system the unit-cell parameters showed high non-isomorphism. These packing variations were exploited in order to conduct a systematic study of conformational changes in PfDTD. It is shown that the disposition of a ten-residue insertion loop affects packing within the PfDTD crystals and seems to determine the non-isomorphism in unit-cell parameters. By tracking the changes in PfDTD unit cells, it was possible to map conformational differences within PfDTD that may be of significance for enzyme activity. [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] Getting the best out of long-wavelength X-rays: de novo chlorine/sulfur SAD phasing of a structural protein from ATVACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2010Adeline Goulet The structure of a 14,kDa structural protein from Acidianus two-tailed virus (ATV) was solved by single-wavelength anomalous diffraction (SAD) phasing using X-ray data collected at 2.0,Å wavelength. Although the anomalous signal from methionine sulfurs was expected to suffice to solve the structure, one chloride ion turned out to be essential to achieve phasing. The minimal data requirements and the relative contributions of the Cl and S atoms to phasing are discussed. This work supports the feasibility of a systematic approach for the solution of protein crystal structures by SAD based on intrinsic protein light atoms along with associated chloride ions from the solvent. In such cases, data collection at long wavelengths may be a time-efficient alternative to selenomethionine substitution and heavy-atom derivatization. [source] Direct-method SAD phasing of proteins enhanced by the use of intrinsic bimodal phase distributions in the subsequent phase-improvement processACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Li-Jie Wu A modified SAD (single-wavelength anomalous diffraction) phasing algorithm has been introduced in the latest version of the program OASIS. In addition to direct-method phases and figures of merit, Hendrickson,Lattman coefficients that correspond to the original unresolved bimodal phase distributions are also output and used in subsequent phase-improvement procedures in combination with the improved phases. This provides the possibility of rebreaking the SAD phase ambiguity using the ever-improving phases resulting from the phase-improvement process. Tests using experimental SAD data from six known proteins showed that in all cases the new treatment produced significant improved results. [source] Characterization of gadolinium complexes for SAD phasing in macromolecular crystallography: application to CbpFACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Rafael Molina Seven Gd complexes were used in the preparation of heavy-atom derivatives for solving the structure of choline-binding protein F (CbpF), a 36,kDa surface protein from Streptococcus pneumoniae, by the SAD method. CbpF was used as a model system to analyse the phasing capability of each of the derivatives. Three different aspects have been systematically characterized: the efficacy of cocrystallization versus soaking in the binding of the different Gd complexes, their mode of interaction and a comparative study of SAD phasing using synchrotron radiation and using a rotating-anode generator. This study reveals the striking potential of these complexes for SAD phasing using a laboratory source and further reinforces their relevance for high-throughput macromolecular crystallography. [source] Structure of a fatty acid-binding protein from Bacillus subtilis determined by sulfur-SAD phasing using in-house chromium radiationACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Jie Nan Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5,Å resolution using Cr,K, radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family. [source] EDM,DEDM and protein crystal structure solutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Rocco Caliandro Electron-density modification (EDM) procedures are the classical tool for driving model phases closer to those of the target structure. They are often combined with automated model-building programs to provide a correct protein model. The task is not always performed, mostly because of the large initial phase error. A recently proposed procedure combined EDM with DEDM (difference electron-density modification); the method was applied to the refinement of phases obtained by molecular replacement, ab initio or SAD phasing [Caliandro, Carrozzini, Cascarano, Giacovazzo, Mazzone & Siliqi (2009), Acta Cryst. D65, 249,256] and was more effective in improving phases than EDM alone. In this paper, a novel fully automated protocol for protein structure refinement based on the iterative application of automated model-building programs combined with the additional power derived from the EDM,DEDM algorithm is presented. The cyclic procedure was successfully tested on challenging cases for which all other approaches had failed. [source] De novo sulfur SAD phasing of the lysosomal 66.3,kDa protein from mouseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Kristina Lakomek The 66.3,kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75,kDa preproprotein which is further processed into 28 and 40,kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3,kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3,kDa variant and the double-chain form consisting of a 28,kDa fragment and a 40,kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3,kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9,Å on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4,Å using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an Rfree factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks. [source] Using 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] Solution of protein crystallographic structures by high-pressure cryocooling and noble-gas phasingACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2006Chae Un Kim Room-pressure flash-cryocooling of protein crystals is the standard way to reduce radiation damage during data collection. Typically, it is necessary to find cryoprotection conditions by trial and error, a process that is not always successful. Recently, a new method, high-pressure cryocooling, was developed that does not require penetrative cryoprotectants and typically yields very high quality diffraction. Since this method involves helium gas as a pressurizing medium, it was of great interest to see whether the method could be extended to diffraction phasing by the incorporation of heavy noble gases such as krypton. A modified Kr,He high-pressure cyrocooling procedure is described wherein crystals are first pressurized with krypton gas to 10,MPa for 1,h. The krypton pressure is then released and the crystals are repressurized with helium over 150,MPa and cooled to liquid-nitrogen temperatures. Porcine pancreas elastase (PPE; 240 residues, 26,kDa) was selected as a test case for this study. Excellent diffraction was achieved by high-pressure cryocooling without penetrating cryoprotectants. A single 0.31 occupied krypton site in a PPE molecule [Bijvoet amplitude ratio (,|,F|,/,F,) of 0.53%] was successfully used for SAD phasing at 1.3,Å. This method has the potential to greatly simplify obtaining protein structures. [source] Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting methodACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2005Yu Kitago A novel and easy crystal-mounting technique was developed for the sulfur SAD method using Cr,K, radiation (2.29,Å). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection in order to eliminate their X-ray absorption. The superiority and reproducibility of the data sets with this mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of Cr,K, radiation, the anomalous signal ,|,F|,/,|F|, of PH1109 is expected to be 1.72% as this protein of 144 residues includes four methionines and two cysteines. Sulfur SAD phasing was performed using SHELXD and SHELXE. In the case of the data set obtained using this novel crystal-mounting technique, 54.9% of all residues were built with side chains automatically by RESOLVE. On the other hand, only 16.0% were built with side chains for the data set collected using the standard cryoloop. These results indicated that this crystal-mounting technique was superior to the standard loop-mounting method for the measurement of small anomalous differences at longer wavelength and yielded better results in sulfur-substructure solution and initial phasing. The present study demonstrates that the sulfur SAD method with a chromium source becomes enhanced and more practical for macromolecular structure determination using the new crystal-mounting technique. [source] Going soft and SAD with manganeseACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2005Paula S. Salgado SAD phasing has been revisited recently, with experiments being carried out using previously unconventional sources of anomalous signal, particularly lighter atoms and softer X-rays. A case study is reported using the 75,kDa RNA-dependent RNA polymerase of the bacteriophase ,6, which binds a Mn atom and crystallizes with three molecules in the asymmetric unit. X-ray diffraction data were collected at a wavelength of 1.89,Å and although the calculated anomalous signal from the three Mn atoms was only 1.2%, SHELXD and SOLVE were able to locate these atoms. SOLVE/RESOLVE used this information to obtain SAD phases and automatically build a model for the core region of the protein, which possessed the characteristic features of the right-hand polymerase motif. These results demonstrate that with modern synchrotron beamlines and software, manganese phasing is a practical tool for solving the structure of large proteins. [source] SAD phasing by combination of direct methods with the SOLVE/RESOLVE procedure.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Erratum In the paper by Wang et al. (2004) an error in equation (18) was published. The correct version of this equation is given below. The following acknowledgement was also missing from the paper. This work is supported by the Innovation Project of the Chinese Academy of Sciences and the 973 Project (Grant Nos. G1999075604 and 2002CB713801) of the Ministry of Science and Technology of China. [source] Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasingACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003J. Shaun Lott The protein PAE2307 is a member of a protein family of unknown function which is conserved among a number of bacterial and archaeal species. The protein was overexpressed in Escherichia coli, purified and crystallized in two crystal forms. The prevalent form was twinned, but the other diffracted to 1.45,Å resolution. The non-twinned crystals proved difficult to reproduce, so screening of potential heavy-atom derivatives by native polyacrylamide gel electrophoresis was used to establish suitable derivatization conditions. This process enabled the production of a K2Pt(NO2)4 derivative that was used to collect a single-wavelength anomalous diffraction (SAD) data set from the only available crystal. Phase information of high quality was obtained, enabling the calculation of an interpretable electron-density map. [source] Crystallization and calcium/sulfur SAD phasing of the human EF-hand protein S100A2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Michael Koch Human S100A2 is an EF-hand protein and acts as a major tumour suppressor, binding and activating p53 in a Ca2+ -dependent manner. Ca2+ -bound S100A2 was crystallized and its structure was determined based on the anomalous scattering provided by six S atoms from methionine residues and four calcium ions present in the asymmetric unit. Although the diffraction data were recorded at a wavelength of 0.90,Å, which is usually not assumed to be suitable for calcium/sulfur SAD, the anomalous signal was satisfactory. A nine-atom substructure was determined at 1.8,Å resolution using SHELXD, and SHELXE was used for density modification and phase extension to 1.3,Å resolution. The electron-density map obtained was well interpretable and could be used for automated model building by ARP/wARP. [source] Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of a human condensin SMC2 hinge domain with short coiled coilsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Kazuki Kawahara In higher eukaryotes, the condensin complex, which mainly consists of two structural maintenance of chromosomes (SMC) subunits, SMC2 (CAP-E) and SMC4 (CAP-C), plays a critical role in the formation of higher order chromosome structures during mitosis. Biochemical and electron-microscopic studies have revealed that the SMC2 and SMC4 subunits dimerize through the interaction of their hinge domains, forming a characteristic V-shaped heterodimer. However, the details of their function are still not fully understood owing to a lack of structural information at the atomic level. In this study, the human SMC2 hinge domain with short coiled coils was cloned, expressed, purified and crystallized in the orthorhombic space group C222 in native and SeMet-derivatized forms. Because of the poor diffraction properties of these crystals, the mutant Leu68,SeMet was designed and crystallized in order to obtain the experimental phases. The SeMet-derivatized crystals of the mutant belonged to space group P3212, with unit-cell parameters a = b = 128.8, c = 91.4,Å. The diffraction data obtained from a crystal that diffracted to 2.4,Å resolution were suitable for SAD phasing. [source] The purification, crystallization and preliminary diffraction of a glycerophosphodiesterase from Enterobacter aerogenesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2006Colin J. Jackson The metallo-glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) has been cloned, expressed in Escherichia coli and purified. Initial screening of crystallization conditions for this enzyme resulted in the identification of needles from one condition in a sodium malonate grid screen. Removal of the metals from the enzyme and subsequent optimization of these conditions led to crystals that diffracted to 2.9,Å and belonged to space group P213, with unit-cell parameter a = 164.1,Å. Self-rotation function analysis and VM calculations indicated that the asymmetric unit contains two copies of the monomeric enzyme, corresponding to a solvent content of 79%. It is intended to determine the structure of this protein utilizing SAD phasing from transition metals or molecular replacement. [source] | ||||||||||