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Heavy-atom Derivatives (heavy-atom + derivative)

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Chemistry100%

Selected Abstracts

Heavy-atom derivatives in lipidic cubic phases: results on hen egg-white lysozyme tetragonal derivative crystals with Gd-HPDO3A complex

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004
Éric Girard
Gd-HPDO3A, a neutral gadolinium complex, is a good candidate for obtaining heavy-atom-derivative crystals by the lipidic cubic phase crystallization method known to be effective for membrane proteins. Gadolinium-derivative crystals of hen egg-white lysozyme were obtained by co-crystallizing the protein with 100,mM Gd-HPDO3A in a monoolein cubic phase. Diffraction data were collected to a resolution of 1.7 Å using Cu,K, radiation from a rotating-anode generator. Two binding sites of the gadolinium complex were located from the strong gadolinium anomalous signal. The Gd-atom positions and their refined occupancies were found to be identical to those found in derivative crystals of hen egg-white lysozyme obtained by co-crystallizing the protein with 100,mM Gd-HPDO3A using the hanging-drop technique. Moreover, the refined structures are isomorphous. The lipidic cubic phase is not disturbed by the high concentration of Gd-­HPDO3A. This experiment demonstrates that a gadolinium complex, Gd-HPDO3A, can be used to obtain derivative crystals by the lipidic cubic phase crystallization method. Further studies with membrane proteins that are known to crystallize in lipidic cubic phases will be undertaken with Gd-HPDO3A and other Gd complexes to test whether derivative crystals with high Gd-site occupancies can be obtained. [source]

5-Amino-2,4,6-tribromoisophthalic acid: the MAD triangle for experimental phasing

ACTA CRYSTALLOGRAPHICA SECTION C, Issue 5 2009
Tobias Beck
The title compound, C8H4Br3NO4, shows an extensive hydrogen-bond network. In the crystal structure, molecules are linked into chains by COO,H...O bonds, and pairs of chains are connected by additional COO,H...O bonds. This chain bundle shows stacking interactions and weak N,H...O hydrogen bonds with adjacent chain bundles. The three Br atoms present in the molecule form an equilateral triangle. This can be easily identified in the heavy-atom substructure when this compound is used as a heavy-atom derivative for experimental phasing of macromolecules. The title compound crystallizes as a nonmerohedral twin. [source]

Using barium ions for heavy-atom derivatization and phasing of xylanase II from Trichoderma longibrachiatum

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2007
Natalia 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]

The structure of l -rhamnulose-1-phosphate aldolase (class II) solved by low-resolution SIR phasing and 20-fold NCS averaging

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2002
Markus Kroemer
The enzyme l -rhamnulose-1-phosphate aldolase catalyzes the reversible cleavage of l -rhamnulose-1-phosphate to dihydroxyacetone phosphate and l -­lactaldehyde. It is a homotetramer with an Mr of 30,000 per subunit and crystallized in space group P3221. The enzyme shows a low sequence identity of 18% with the structurally known l -fuculose-1-­phosphate aldolase that splits a stereoisomer in a similar reaction. Structure analysis was initiated with a single heavy-atom derivative measured to 6,Å resolution. The resulting poor electron density, a self-rotation function and the working hypothesis that both enzymes are C4 symmetric with envelopes that resemble one another allowed the location of the 20 protomers of the asymmetric unit. The crystal-packing unit was a D4 -symmetric propeller consisting of five D4 -­symmetric octamers around an internal crystallographic twofold axis. Presumably, the propellers associate laterally in layers, which in turn pile up along the 32 axis to form the crystal. The non-crystallographic symmetry was used to extend the phases to the 2.7,Å resolution limit and to establish a refined atomic model of the enzyme. The structure showed that the two enzymes are indeed homologous and that they possess chemically similar active centres. [source]

Crystallization and preliminary X-ray analysis of the selenate reductase from Thauera selenatis

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002
Megan J. Maher
Selenate reductase from Thauera selenatis was crystallized using ammonium sulfate as a precipitant. Crystals of selenate reductase belong to the space group C2, with unit-cell parameters a = 116.9, b = 67.5, c = 186.7,Å, , = 90°. Native data to 2.1,Å resolution have been collected and a heavy-atom derivative has been identified following soaking of the crystals in a solution of trimethyl lead acetate. [source]

Features of the secondary structure of a protein molecule from powder diffraction data

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010
Sebastian Basso
Protein powder diffraction is shown to be suitable for obtaining de novo solutions to the phase problem at low resolution via phasing methods such as the isomorphous replacement method. Two heavy-atom derivatives (a gadolinium derivative and a holmium derivative) of the tetragonal form of hen egg-white lysozyme were crystallized at room temperature. Using synchrotron radiation, high-quality powder patterns were collected in which pH-induced anisotropic lattice-parameter changes were exploited in order to reduce the challenging and powder-specific problem of overlapping reflections. The phasing power of two heavy-atom derivatives in a multiple isomorphous replacement analysis enabled molecular structural information to be obtained up to approximately 5.3,Å resolution. At such a resolution, features of the secondary structure of the lysozyme molecule can be accurately located using programs dedicated to that effect. In addition, the quoted resolution is sufficient to determine the correct hand of the heavy-atom substructure which leads to an electron-density map representing the protein molecule of proper chirality. [source]

A rational approach to heavy-atom derivative screening

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010
M. Gordon Joyce
Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the `classical' heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom compound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the process of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing. [source]

Characterization of gadolinium complexes for SAD phasing in macromolecular crystallography: application to CbpF

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009
Rafael 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]

Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasing

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003
J. 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 preliminary X-ray study of recombinant betaine,homocysteine S -­methyltransferase from rat liver

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2002
Beatriz González
Betaine,homocysteine S -methyltransferase is one of the three enzymes involved in homocysteine catabolism. It uses betaine as the methyl donor to convert homocysteine into methionine, also producing dimethylglycine. Recombinant BHMT from rat liver was crystallized by the vapour-diffusion method in both native and seleniomethionyl-labelled forms. Crystals belong to space group P21, with unit-cell parameters a = 57.8, b = 149.3, c = 96.2,Å, , = 92.9°. Data from native, seleniomethionine-labelled and two heavy-atom derivatives were collected using synchrotron sources. Self-rotation function and sedimentation-velocity experiments suggest that the enzyme is tetrameric with 222 symmetry. [source]

Generating isomorphous heavy-atom derivatives by a quick-soak method.

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2002
Part I: test cases
Screening for heavy-atom derivatives remains a time-consuming and cumbersome process that often results in non-isomorphous derivatives whose phases cannot be combined. Using lysozyme and Fc,RIII receptor crystals as test cases, an improved soaking method for the generation of conventional heavy-atom derivatives has been developed. The method is based on soaking crystals in heavy-atom compounds for a very brief time at near-saturation concentrations. Compared with the current heavy-atom soaking method, which often takes days to achieve a derivatization, the quick-soak method completes a derivatization within 10,min to 2,h. The bound heavy-atom sites display higher peak heights from quick soaks than from overnight soaks in all cases tested. The quick-soak derivatives also preserved native-like diffraction resolution and data quality that was better than the prolonged-soak derivatives. Furthermore, derivatives generated by brief soaks are more isomorphous to the native than those generated by overnight soaks. Short soaks not only increase the likelihood of success in heavy-atom screening by reducing the pitfalls associated with prolonged soaks, such as lack of isomorphism and overall lattice disorder, but also have the potential to transform a time-consuming derivative screening into an `on-the-fly' real-time derivatization process. [source]

Generating isomorphous heavy-atom derivatives by a quick-soak method.

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2002
Part II: phasing of new structures
A quick-soak method has been applied to generate de novo heavy-atom phasing to solve two new protein structures, a type II transforming growth factor , receptor (TBRII) and a natural killer cell receptor,ligand complex, NKG2D,ULBP3. In the case of TBRII, a crystal derivatized for only 10,min in saturated HgCl2 provided adequate phasing for structure determination. Comparison between HgCl2 derivatives generated by 10,min soaking and by 12,h soaking revealed similar phasing statistics. The shorter soak, however, resulted in a derivative more isomorphous to the native than the longer soak as judged by changes in the unit-cell parameter a upon derivatization as well as by the quality of a combined SIRAS electron-density map. In the case of the NKG2D,ULBP3 structure, all overnight soaks in heavy-atom solutions resulted in crystal lattice disorder and only the quick soaks preserved diffraction. Despite fragile lattice packing, the quick-soaked K2PtCl4 derivative was isomorphous with the native crystal and the electron-density map calculated from combined SIR and MAD phases is better than that calculated from MAD phases alone. Combined with mass-spectrometry-assisted solution heavy-atom derivative screening and the use of synchrotron radiation, the quick-soak derivatization has the potential to transform the time-consuming conventional heavy-atom search into a real-time `on-the-fly' derivatization process that will benefit high-throughput structural genomics. [source]

Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with tetragonal hen egg-white lysozyme

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002
Éric 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]

A novel 40,kDa protein from goat mammary secretions: purification, crystallization and preliminary X-ray diffraction studies

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001
P. Kumar
A novel 40,kDa protein has been purified from dry secretions of the mammary gland of goats. The first 15 N-terminal residues were sequenced and showed a sequence identity of 30% to a novel 39,kDa whey protein from bovine mammary secretions. The protein was crystallized by the microdialysis method. Protein was dissolved to a concentration of 40,mg,ml,1 in 0.025,M Tris,HCl pH 8.0 and equilibrated with the same buffer containing 19%(v/v) ethanol. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 66.1, b = 107.8, c = 63.2,Å and one molecule per asymmetric unit. Intensity data were collected to 2.9,Å resolution, with a completeness of 95%. Since no similar model is available in the protein structure database, heavy-atom derivatives have been prepared and three-dimensional structure determination using the isomorphous replacement method is in progress. [source]

Purification, crystallization and identification by X-­ray analysis of a prostate kallikrein from horse seminal plasma

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001
Ana L. Carvalho
The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42,Å resolution were collected at the ESRF synchrotron-radiation source. [source]

Crystallization of agGST1-6, a recombinant glutathione S -transferase from a DDT-resistant strain of Anopheles gambiae

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2001
Pamela H. Roberts
Glutathione S -transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cell. GST activity is elevated in many insecticide-resistant insects, including the DDT-resistant malaria vector Anopheles gambiae. Crystals of the recombinant form of a GST from A. gambiae, agGST1-6, have been grown in at least five different crystal forms, with a broad range of diffraction resolution limits. A complete 2.0,Å data set has been collected on a C -centered orthorhombic crystal form with unit-cell parameters a = 99.0, b = 199.4, c = 89.6,Å. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement. [source]

Crystallization of the cytotoxic domain of a ribosome-inactivating colicin in complex with its immunity protein

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2000
Stephen Carr
The complex between the ribonuclease domain of the ribosome-inactivating colicin E3 and its protein inhibitor, the cognate immunity Im3, has been crystallized and preliminary X-ray characterization has been performed. Single crystals of the 1:1 complex were grown from hanging-drop vapour-diffusion experiments using 2-propanol as a precipitant. The space group is P3121 or P3221, with unit-cell parameters a = b = 93.7, c = 76.2,Å. When cryocooled, these crystals diffract to a resolution of 2.4,Å. A search for suitable conventional heavy-atom derivatives was unsuccessful and so Im3 mutants containing engineered cysteine or methionine residues have been produced for mercury soaks and selenomethionine-labelling experiments, respectively. [source]

Crystallization and preliminary crystallographic analysis of recombinant VSP1 from Arabidopsis thaliana

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010
Zhu-Bing Shi
VSP1 is a defence protein in Arabidopsis thaliana that may also be involved in control of plant development. The recombinant protein has been overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal diffracted to 1.9,Å resolution and a complete X-ray data set was collected at 100,K using Cu,K, radiation from a rotating-anode X-ray source. The crystals belonged to space group C2. As there are no related structures that could be used as a search model for molecular replacement, work is in progress on experimental phasing using heavy-atom derivatives and selenomethionine derivatives. [source]

Purification, crystallization and preliminary crystallographic studies of the complex of interferon-,1 with its receptor

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010
Eugenia Magracheva
Human interferon-,1 (IFN-,1Ins) and the extracellular domain of interferon-,1 receptor (IFN-,1R1) were expressed in Drosophila S2 cells and purified to homogeneity. Both IFN-,1Ins and interferon-,1 produced from Escherichia coli (IFN-,1Bac) were coupled with IFN-,1R1 at room temperature and the complexes were purified by gel filtration. Both complexes were crystallized; the crystals were flash-frozen at 100,K and diffraction data were collected to 2.16 and 2.1,Å, respectively. Although the IFN-,1Bac,IFN-,1R1 and IFN-,1Ins,IFN-,1R1 complexes differed only in the nature of the expression system used for the ligand, their crystallization conditions and crystal forms were quite different. A search for heavy-atom derivatives as well as molecular-replacement trials are in progress. [source]

Crystallization and preliminary X-ray diffraction analysis of inositol 1,3,4,5,6-pentakisphosphate kinase from Arabidopsis thaliana

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010
Jose Ignacio Baños-Sanz
Inositol 1,3,4,5,6-pentakisphosphate kinase (IP5 2-K) is an enzyme involved in inositol metabolism that synthesizes IP6 (inositol 1,2,3,4,5,6-hexakisphosphate) from inositol 1,3,4,5,6-pentakisphosphate (IP5) and ATP. IP6 is the major phosphorus reserve in plants, while in mammals it is involved in multiple cellular events such as DNA editing and chromatin remodelling. In addition, IP6 is the precursor of other highly phosphorylated inositols which also play highly relevant roles. IP5 2-K is the only enzyme that phosphorylates the 2-OH axial position of the inositide and understanding its molecular mechanism of substrate specificity is of great interest in cell biology. IP5 2-K from Arabidopsis thaliana has been expressed in Escherichia coli as two different fusion proteins and purified. Both protein preparations yielded crystals of different quality, always in the presence of IP6. The best crystals obtained for X-ray crystallographic analysis belonged to space group P212121, with unit-cell parameters a = 58.124, b = 113.591, c = 142.478,Å. Several diffraction data sets were collected for the native enzyme and two heavy-atom derivatives using a synchrotron source. [source]

Crystallization and preliminary X-ray analysis of the HA3 component of Clostridium botulinum type C progenitor toxin

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2007
Toshio Nakamura
HA3, a 70,kDa haemagglutinating protein, is a precursor form of HA3a and HA3b, the subcomponents of Clostridium botulinum type C 16S progenitor toxin. In this report, recombinant HA3 protein was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.6,Å resolution and the crystal belonged to the hexagonal space group P63. Matthews coefficient and self-rotation function calculations indicate that there is probably one molecule of HA3 in the asymmetric unit. A search for heavy-atom derivatives has been undertaken. [source]