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Anomalous Scattering (anomalous + scattering)
Terms modified by Anomalous Scattering 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] 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] Teaching crystallography to undergraduate physical chemistry studentsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5-2 2010Virginia B. Pett Teaching goals, laboratory experiments and homework assignments are described for teaching crystallography as part of two undergraduate physical chemistry courses. A two-week teaching module is suggested for introductory physical chemistry, including six to eight classroom sessions, several laboratory experiences and a 3,h computer-based session, to acquaint undergraduate physical chemistry students with crystals, diffraction patterns, the mathematics of structure determination by X-ray diffraction, data collection, structure solution and the chemical insights available from crystal structure information. Student projects and laboratory work for three to four weeks of an advanced physical chemistry course are presented. Topics such as symmetry operators, space groups, systematic extinctions, methods of solving the phase problem, the Patterson map, anomalous scattering, synchrotron radiation, crystallographic refinement, hydrogen bonding and neutron diffraction all lead to the goal of understanding and evaluating a crystallographic journal article. Many of the ideas presented here could also be adapted for inorganic chemistry courses. [source] Instrument-independent specification of the diffraction geometry and polarization state of the incident X-ray beamJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 1 2009Marc Schiltz This work augments the proposal of Schwarzenbach & Flack [J. Appl. Cryst. (1989), 22, 601,605], who have advocated the use of a diffractometer-independent definition of the azimuthal angle , to specify the diffraction geometry of a Bragg reflection. It is here proposed that one additional angle ,, which is also based on a diffractometer-independent definition, is needed to encode the direction of linear polarization for those experiments where this quantity is of importance. This definition is then extended to the cases of partially and/or elliptically polarized X-ray beams, and the use of three normalized Stokes parameters, P1, P2 and P3, together with ,, is advocated in order to characterize exhaustively the polarization state of the incident beam. The conventions proposed here present a general, unambiguous and economical means of encoding the information about the diffraction geometry, without the need to record any further information about the instrument, crystal orientation matrix and goniometer angles. Data-processing software using these definitions to analyse polarization-dependent phenomena becomes instrument-independent and completely general. These methods have been implemented in the macromolecular phasing program SHARP for exploiting the polarization anisotropy of anomalous scattering in protein crystals. [source] Phasing possibilities using different wavelengths with a xenon derivativeJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2 2002Santosh Panjikar Xenon derivatives are generally expected to be isomorphous with the native; however, the K - and L -absorption edges are not easily accessible on most synchrotron beamlines, which might limit their usefulness in phase determination. Various phasing procedures for xenon-derivatized porcine pancreatic elastase have been investigated using data sets measured at three generally accessible wavelengths. The importance of highly redundant data in measuring precise anomalous differences is highlighted and it is shown that, after such measurements, a single isomorphous replacement anomalous scattering (SIRAS) procedure yields a better phase set than those generated by single anomalous scattering (SAS) or multiwavelength anomalous diffraction (MAD) procedures. [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] The application of eigensymmetries of face forms to anomalous scattering and twinning by merohedry in X-ray diffractionACTA CRYSTALLOGRAPHICA SECTION A, Issue 3 2010H. Klapper The face form (crystal form) {hkl} which corresponds to an X-ray reflection hkl is considered. The eigensymmetry (inherent symmetry) of such a face form can be used to derive general results on the intensities of the corresponding X-ray reflections. Two cases are treated. (i) Non-centrosymmetric crystals exhibiting anomalous scattering: determination of reflections hkl for which Friedel's rule is strictly valid, i.e.I(hkl) = I() (Friedel pair, centric reflection), or violated, i.e.I(hkl) , I() (Bijvoet pair, acentric reflection). It is shown that those reflections hkl strictly obey Friedel's rule, for which the corresponding face form {hkl} is centrosymmetric. If the face form {hkl} is non-centrosymmetric, Friedel's rule is violated due to anomalous scattering. (ii) Crystals twinned by merohedry: determination of reflections hkl, the intensities of which are affected (or not affected) by the twinning. It is shown that the intensity is affected if the twin element is not a symmetry element of the eigensymmetry of the corresponding face form {hkl}. The intensity is not affected if the twin element belongs to the eigensymmetry of {hkl} (`affected' means that the intensities of the twin-related reflections are different for different twin domain states owing to differences either in geometric structure factors or in anomalous scattering or in both). A simple procedure is presented for the determination of these types of reflections from Tables 10.1.2.2 and 10.1.2.3 of International Tables for Crystallography, Vol. A [Hahn & Klapper (2002). International Tables for Crystallography, Vol. A, Part 10, edited by Th. Hahn, 5th ed. Dordrecht: Kluwer]. The application to crystal-structure determination of crystals twinned by merohedry (reciprocal space) and to X-ray diffraction topographic mapping of twin domains (direct space) is discussed. Relevant data and twinning relations for the 63 possible twin laws by merohedry in the 26 merohedral point groups are presented in Appendices A to D. [source] Structure of the N-terminal fragment of Escherichia coli Lon proteaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Mi Li The structure of a recombinant construct consisting of residues 1,245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6,Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal ,-helix. The structure of the first subdomain (residues 1,117), which consists mostly of ,-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates. [source] A dipicolinate lanthanide complex for solving protein structures using anomalous diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Guillaume Pompidor Tris-dipicolinate lanthanide complexes were used to prepare derivative crystals of six proteins: hen egg-white lysozyme, turkey egg-white lysozyme, thaumatin from Thaumatococcus daniellii, urate oxidase from Aspergillus flavus, porcine pancreatic elastase and xylanase from Trichoderma reesei. Diffraction data were collected using either synchrotron radiation or X-rays from a laboratory source. In all cases, the complex turned out to be bound to the protein and the phases determined using the anomalous scattering of the lanthanide led to high-quality electron-density maps. The binding mode of the complex was characterized from the refined structures. The lanthanide tris-dipicolinate was found to bind through interactions between carboxylate groups of the dipicolinate ligands and hydrogen-bond donor groups of the protein. In each binding site, one enantiomeric form of the complex is selected from the racemic solution according to the specific site topology. For hen egg-white lysozyme and xylanase, derivative crystals obtained by cocrystallization belonged to a new monoclinic C2 crystal form that diffracted to high resolution. [source] `Broken symmetries' in macromolecular crystallography: phasing from unmerged dataACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Marc Schiltz The space-group symmetry of a crystal structure imposes a point-group symmetry on its diffraction pattern, giving rise to so-called symmetry-equivalent reflections. Instances in macromolecular crystallography are discussed in which the symmetry in reciprocal space is broken, i.e. where symmetry-related reflections are no longer equivalent. Such a situation occurs when the sample suffers from site-specific radiation damage during the X-ray measurements. Another example of broken symmetry arises from the polarization anisotropy of anomalous scattering. In these cases, the genuine intensity differences between symmetry-related reflections can be exploited to yield phase information in the structure-solution process. In this approach, 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 effects are explicitly modelled and refined and become a source of supplementary phase information. [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] 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] 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] 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] What happens when the signs of anomalous differences or the handedness of substructure are inverted?ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2007Jiawei Wang Proper solution of a macromolecular crystal structure based on anomalous scattering and/or isomorphous differences requires that the anomalous differences in reflection amplitudes be measured properly and that the correct enantiomer of the substructure be selected. If this information is wrong then the resulting electron-density maps will not show the correct structural features, but the reflection phases and map features will be related to the correct ones in a specific way. This text aims to explain how misinterpretation of the Bijvoet differences or of the substructure affects the resulting phases and electron-density maps. [source] Radiation-induced site-specific damage of mercury derivatives: phasing and implicationsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2005Udupi A. Ramagopal The behavior of mercury-derivatized triclinic crystals of a 60,kDa protein target from the New York Structural GenomiX Research Consortium provides novel insights into the mechanism of heavy-atom-specific radiation damage and its potential exploitation for de novo structure solution. Despite significant anomalous signal, structure solution by classic SAD and MAD phasing approaches was not successful. A detailed analysis revealed that significant isomorphic variation of the diffracted intensities was induced by X-ray irradiation. These intensity changes allowed the crystal structure to be solved by the radiation-damage-induced phasing (RIP) technique. Inspection of the crystal structure and electron-density maps demonstrated that the covalent S,Hg bonds at all four derivatized cysteine sites were much more susceptible to radiation-induced cleavage than other bonds typically present in native proteins. A simple diagnostic is described to identify the fingerprint of such decay at the time of data collection/processing. The rapid radiation-induced decomposition of mercury adducts is consistent with the difficulties frequently associated with the experimental phasing of mercury derivatives and suggests a straightforward solution to overcome this limitation by radiation-damage-induced phasing with anomalous scattering (RIPAS). These results indicate that historically recalcitrant and newly emerging difficulties associated with Hg phasing should be revisited. [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] 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] Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with tetragonal hen egg-white lysozymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002Éric Girard A neutral gadolinium complex, Gd-HPDO3A, is shown to be a good candidate to use to obtain heavy-atom derivatives and solve macromolecular structures using anomalous dispersion. Tetragonal crystals of a gadolinium derivative of hen egg-white lysozyme were obtained by co-crystallization using different concentrations of the complex. Diffraction data from three derivative crystals (100, 50 and 10,mM) were collected to a resolution of 1.7,Å using Cu,K, radiation from a rotating anode. Two strong binding sites of the gadolinium complex to the protein were located from the gadolinium anomalous signal in both the 100 and 50,mM derivatives. A single site is occupied in the 10,mM derivative. Phasing using the anomalous signal at a single wavelength (SAD method) leads to an electron-density map of high quality. The structure of the 100,mM derivative has been refined. Two molecules of the gadolinium complex are close together. Both molecules are located close to tryptophan residues. Four chloride ions were found. The exceptional quality of the SAD electron-density map, only enhanced by solvent flattening, suggests that single-wavelength anomalous scattering with the Gd-HPDO3A complex may be sufficient to solve protein structures of high molecular weight by synchrotron-radiation experiments, if not by laboratory experiments. [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] 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] 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] Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe2+ metal-ion preferenceACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2008Colin J. Jackson The structure of a malonate-bound form of the glycerophosphodiesterase from Enterobacter aerogenes, GpdQ, has been refined at a resolution of 2.2,Å to a final R factor of 17.1%. The structure was originally solved to 2.9,Å resolution using SAD phases from Zn2+ metal ions introduced into the active site of the apoenzyme [Jackson et al. (2007), J. Mol. Biol.367, 1047,1062]. However, the 2.9,Å resolution was insufficient to discern significant details of the architecture of the binuclear metal centre that constitutes the active site. Furthermore, kinetic analysis revealed that the enzyme lost a significant amount of activity in the presence of Zn2+, suggesting that it is unlikely to be a catalytically relevant metal ion. In this communication, a higher resolution structure of GpdQ is presented in which malonate is visibly coordinated in the active site and analysis of the native metal-ion preference is presented using atomic absorption spectroscopy and anomalous scattering. Catalytic implications of the structure and its Fe2+ metal-ion preference are discussed. [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] Molecular homochirality and the parity-violating energy difference.CHIRALITY, Issue 2 2008A critique with new proposals Abstract Previous proposals for the origin of molecular homochirality, based on the effect of the weak neutral current (WNC) on enantiomers, and the amplification of the resultant parity-violating energy difference (PVED), are possibly flawed. The additive amplification of PVED in crystals and polymers ("Yamagata hypothesis") cannot lead to detectable levels of optical activity, the original theory apparently overestimating PVED by a factor equal to Avogadro's number. An alternative theory based on the irreversible and spontaneous evolution of a dynamically fluctuating system is apparently impractical. However, the nonlinear amplification of PVED via autocatalytic polymerization may be possible as indicated by a simplified physico-chemical approach. This may also occur during crystallization and melting, and form the basis of the second order asymmetric transformation. (Thus, reported differences in the melting points of enantiomers in several cases may well be real). Also, the preponderance of racemic compounds over conglomerates may be based on the destabilization of the conglomerate by the action of the WNC on the crystalline lattice. The WNC may also be involved in the anomalous scattering of X-rays, which possibly arises from their circular polarization: the current theory would need to be revised accordingly. Chirality, 2008. © 2007 Wiley-Liss, Inc. [source] | ||||||||||