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Isomorphous Replacement (isomorphous + replacement)
Kinds of Isomorphous Replacement Selected AbstractsOverview and new developments in softer X-ray (2Å < , < 5Å) protein crystallographyJOURNAL OF SYNCHROTRON RADIATION, Issue 1 2004John R. Helliwell New methodologies with synchrotron radiation and X-ray free electron lasers (XFELs) in structural biology are being developed. Recent trends in harnessing softer X-rays in protein crystallography for phase determination are described. These include reference to a data-collection test at 2.6 Å wavelength with a lysozyme crystal on SRS station 7.2 (Helliwell, 1983) and also use of softer X-rays (2,Å wavelength) to optimise f," at the xenon L1 absorption edge in the Single Isomorphous Replacement Optimised Anomalous Scattering ('SIROAS') structure determination of apocrustacyanin A1 with four, partially occupied, xenon atoms (Cianci et al., 2001; Chayen et al., 2000). The hand of the protein was determined using the f," enhanced sulphur anomalous signal from six disulphides in the protein dimer of 40,kDa. In a follow-up study the single wavelength xenon L1 -edge f," optimised data set alone was used for phase determination and phase improvement by solvent flattening etc. (CCP4 DM) (Olczak et al., 2003). Auto-tracing of the protein was feasible but required additional diffraction data at higher resolution. This latter could be avoided in future by using improved tilted detector settings during use of softer X-rays, i.e. towards back-scattering recording (Helliwell, 2002). The Olczak et al. study has already led to optimisation of the new SRS beamline MPW,MAD,10 (see www.nwsgc.ac.uk) firstly involving the thinning of the beryllium windows as much as possible and planning for a MAR Research tilted detector `desk top beamline' geometry. Thus the use of softer, i.e. 2 to 3,Å wavelength range, X-rays will allow optimisation of xenon and iodine L -edge f," and enhancing of sulphur f," signals for higher throughput protein crystallography. Softer X-rays utilisation in protein crystallography includes work done on SRS bending-magnet station 7.2 in the early 1980s by the author as station scientist (Helliwell, 1984). In the future development of XFELs these softer X-ray wavelengths could also be harnessed and relax the demands to some extent on the complexity and cost of an XFEL. Thus, by use of say 4,Å XFEL radiation and use of a back-scattering geometry area detector the single molecule molecular transform could be sampled to a spatial resolution of 2,Å, sufficient, in principle, for protein model refinement (Miao et al., 1999). Meanwhile, Miao et al. (2003) report the first experimental recording of the diffraction pattern from intact Escherichia coli bacteria using coherent X-rays, with a wavelength of 2,Å, at a resolution of 30,nm and a real-space image constructed. The new single-particle X-ray diffraction-imaging era has commenced. [source] Bentonite as a Natural Adsorbent for the Sorption of Iron from the Ground Water Exploited from Aswan Area, EgyptGROUND WATER MONITORING & REMEDIATION, Issue 1 2004Gharib M. Taha Sorption of dissolved Fe2+ on bentonite was studied using a batch technique. The distribution coefficient, Kd, was evaluated for a bentonite-iron system as a function of contact time, pH, sorbent and sorbate concentrations, and temperature. Sorption results were interpreted in terms of Freundlich's and Langmuir's equations. Thermodynamic parameters for the sorption system were determined at three temperatures: 298°, 308°, and 318°K. The values of ,H°(-4.0 kjmol,1) and ,G°(-2.46 Kjmol,1) at 298°K (25°C) suggest that sorption of iron on bentonite is an exothermic and a spontaneous process. The ,G° value became less negative at higher temperatures and, therefore, less iron was sorbed at higher temperatures. The desorption studies with 0.01M CaCl2 and deionized water at iron loading on bentonite showed that more than 90 wt% of the iron is irreversibly sorbed, probably due to the fixation of the iron by isomorphous replacement in the crystal lattice of the sorbent. [source] Anomalous scattering and isomorphous replacement in X-ray diffuse scattering holographyPHYSICA STATUS SOLIDI (A) APPLICATIONS AND MATERIALS SCIENCE, Issue 8 2007Kopecký Abstract Two concepts of X-ray diffuse scattering holography resulting in local atomic structure are presented. The first one uses the anomalous scattering near the absorption edge of a selected element. The second one is based on the variation of atomic scattering factor due to the isomorphous replacement of a selected atom in the structure by another one with different scattering properties. Feasibility of both concepts was demonstrated experimentally on a RbCl single crystal and GaMnAs epitaxial layer. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] A multivariate likelihood SIRAS function for phasing and model refinementACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009Pavol Skubák A likelihood function based on the multivariate probability distribution of all observed structure-factor amplitudes from a single isomorphous replacement with anomalous scattering experiment has been derived and implemented for use in substructure refinement and phasing as well as macromolecular model refinement. Efficient calculation of a multidimensional integration required for function evaluation has been achieved by approximations based on the function's properties. The use of the function in both phasing and protein model building with iterative refinement was essential for successful automated model building in the test cases presented. [source] Structure of the C-terminal domain of nsp4 from feline coronavirusACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Ioannis Manolaridis Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26,31,kb) encodes 15,16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication,transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (,100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8,Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly ,-helical content displaying a unique fold that could be engaged in protein,protein interactions. [source] Twinned crystals and anomalous phasingACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2003Zbigniew Dauter Merohedral or pseudomerohedral twinning of crystals cannot be identified from inspection of the diffraction patterns. Several methods for the identification of twinning and the estimation of the twin fraction are suitable for macromolecular crystals and all are based on the statistical properties of the measured diffraction intensities. If the crystal twin fraction is estimated and is not too close to 0.5, the diffraction data can be detwinned; that is, related to the individual crystal specimen. However, the detwinning procedure invariably introduces additional inaccuracies to the estimated intensities, which substantially increase when the twin fraction approaches 0.5. In some cases, a crystal structure can be solved with the original twinned data by standard techniques such as molecular replacement, multiple isomorphous replacement or multiwavelength anomalous diffraction. Test calculations on data collected from a twinned crystal of gpD, the bacteriophage , capsid protein, show that the single-wavelength anomalous diffraction (SAD) method can be used to solve its structure even if the data set corresponds to a perfectly twinned crystal with a twin fraction of 0.5. [source] MIR phasing using merohedrally twinned crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2003Anke C. Terwisscha van Scheltinga Merohedral twinning is a crystal-growth disorder that seriously hinders the determination of macromolecular crystal structures by isomorphous replacement. The strategies used in the structures solved so far are discussed. Several methods can be used to determine the extent of twinning, the twin fraction and to detwin the data. Accurate determination of the twin fraction by analysing heavy-atom refinement statistics is possible, but only influences the resulting phases slightly. It seems more crucial to restrict the variation in twin fractions between data sets, either by making the twin fractions of some data sets artificially higher or by screening crystals to obtain data with a low twin fraction. [source] Multiple isomorphous replacement on merohedral twins: structure determination of deacetoxycephalosporin C synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2001Anke C. Terwisscha van Scheltinga Merohedral twinning is a packing anomaly that seriously impairs the determination of macromolecular crystal structures. Crystals of deacetoxycephalosporin C synthase (DAOCS), an enzyme involved in the expansion of the penicillin nucleus to form the core structure of the cephalosporin antibiotics, were found to be merohedrally twinned by many diagnostic criteria. Here, the structure determination of DAOCS from twinned crystals based on a combination of isomorphous replacement and the use of a multiple-wavelength diffraction data set is described. [source] Map self-validation: a useful discriminator of phase correctness at low resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2001David A. Langs A new map-validation procedure is based on the correlation-coefficient agreement between the observed structure-factor magnitudes and their extrapolated values from suitably modified electron-density maps from which they have been each in turn systematically excluded. The correlation coefficient tends to a maximum as the phase errors in a map are reduced. This principle was used to resolve the single-wavelength anomalous scattering (SAS) and single-derivative isomorphous replacement (SIR) phase ambiguity for a number of error-free trial structures. Applications employing real data sets tend to be more difficult owing to data incompleteness and errors affecting the construction of the Argand diagram. [source] Structure of XynB, a highly thermostable ,-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2000Andrew A. McCarthy Microorganisms employ a large array of enzymes to break down the cellulose and hemicelluloses of plant biomass. These enzymes, especially those with high thermal stability, have many uses in biotechnology. We have solved the crystal structure of a ,-1,4-xylanase, XynB, from the extremely thermophilic bacterium Dictyoglomus thermophilum, isolate Rt46B.1. The protein crystallized from 1.6,M ammonium sulfate, 0.2,M HEPES pH 7.2 and 10% glycerol, with unit-cell parameters a = b = 91.3, c = 44.9,Å and space group P43. The structure was solved at high resolution (1.8,Å) by X-ray crystallography, using the method of isomorphous replacement with a single mercury derivative, and refined to a final R factor of 18.3% (Rfree = 22.1%). XynB has the single-domain fold typical of family 11 xylanases, comprising a jelly roll of two highly twisted ,-sheets that create a deep substrate-binding cleft. The two catalytic residues, Glu90 and Glu180, occupy this cleft. Compared with other family 11 xylanases, XynB has a greater proportion of polar surface and has a slightly extended C-terminus that, combined with the extension of ,-strand A5, gives additional hydrogen bonding and hydrophobic packing. These factors may account for the enhanced thermal stability of the enzyme. [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] Purification, crystallization and X-ray diffraction analysis of pavine N -methyltransferase from Thalictrum flavumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2008Ankur Jain A cDNA from the plant Thalictrum flavum encoding pavine N -methyltransferase, an enzyme belonging to a novel class of S -adenosylmethionine-dependent N -methyltransferases specific for benzylisoquinoline alkaloids, has been heterologously expressed in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatography and was crystallized in space group P21. The structure was solved at 2.0,Å resolution using a xenon derivative and the single isomorphous replacement with anomalous scattering method. [source] Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2006D. Aragão The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65,Å. The phase problem was solved for a P21 crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P21 has unit-cell parameters a = 105.5, b = 85.7, c = 151.8,Å, , = 105.2°. Model building and refinement are currently under way. [source] Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioidesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2005Marcos Vicente de A. S. Navarro A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293,K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87,Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P212121, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24,Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (VM = 2.5,Å3,Da,1). Iodinated crystals were prepared and a derivative data set was also collected at 2.1,Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5,M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method. [source] Crystallization of the glycogen-binding domain of the AMP-activated protein kinase , subunit and preliminary X-ray analysisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2005Galina Polekhina AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic , subunit and two regulatory subunits, , and ,. Mutations in the , subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the , subunit. Here, the crystallization of GBD in the presence of ,-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein. [source] | ||||||||||