Single-wavelength Anomalous Diffraction Experiment Research Articles

Multivariate estimation of substructure amplitudes for a single-wavelength anomalous diffraction experiment.

To determine a substructure from single-wavelength anomalous diffraction (SAD) data using Patterson or direct methods, the substructure-factor amplitude (|Fa|) is first estimated. Currently, the absolute value of the Bijvoet difference is widely used as an estimate of |Fa| values for SAD data. Here, an equation is derived from multivariate statistics and tested that takes into account the correlation between the observed positive (F+) and negative (F-) Friedel pairs and Fa along with measurement errors in the observed data. The multivariate estimation of |Fa| has been implemented in a new program, Afro. Results on over 180 test cases show that Afro provides a higher correlation to the final substructure-factor amplitudes (calculated from the refined, final substructures) than the Bijvoet differences and improves the robustness of direct-methods substructure detection.

Native SAD phasing at room temperature.

Single-wavelength anomalous diffraction (SAD) is a routine method for overcoming the phase problem when solving macromolecular structures. This technique requires the accurate measurement of intensities to determine differences between Bijvoet pairs. Although SAD experiments are commonly conducted at cryogenic temperatures to mitigate the effects of radiation damage, such temperatures can alter the conformational ensemble of the protein and may impede the merging of data from multiple crystals due to non-uniform freezing. Here, a strategy is presented to obtain high-quality data from room-temperature, single-crystal experiments. To illustrate the strengths of this approach, native SAD phasing at 6.55 keV was used to solve four structures of three model systems at 295 K. The resulting data sets allow automatic phasing and model building, and reveal alternate conformations that reflect the structure of proteins at room temperature.

Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase fromGeobacillus stearothermophilus

Acetylxylan esterases are part of the hemi-cellulolytic system of many microorganisms which utilize plant biomass for growth. Xylans, which are polymeric sugars that constitute a significant part of the plant biomass, are usually substituted with acetyl side groups attached at position 2 or 3 of the xylose backbone units. Acetylxylan esterases hydrolyse the ester linkages of the xylan acetyl groups and thus improve the ability of main-chain hydrolysing enzymes to break down the sugar backbone units. As such, these enzymes play an important part in the hemi-cellulolytic utilization system of many microorganisms that use plant biomass for growth. Interest in the biochemical characterization and structural analysis of these enzymes stems from their numerous potential biotechnological applications. An acetylxylan esterase (Axe2) of this type from Geobacillus stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized. One of the crystal forms obtained (RB1) belonged to the tetragonal space group I422, with unit-cell parameters a = b = 110.2, c = 213.1 Å. A full diffraction data set was collected to 1.85 Å resolution from flash-cooled crystals of the wild-type enzyme at 100 K using synchrotron radiation. A selenomethionine derivative of Axe2 has also been prepared and crystallized for single-wavelength anomalous diffraction experiments. The crystals of the selenomethionine-derivatized Axe2 appeared to be isomorphous to those of the wild-type enzyme and enabled the measurement of a full 1.85 Å resolution diffraction data set at the selenium absorption edge and a full 1.70 Å resolution data set at a remote wavelength. These data are currently being used for three-dimensional structure determination of the Axe2 protein.

C68 from the Sulfolobus islandicus plasmid–virus pSSVx is a novel member of the AbrB-like transcription factor family

The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid-virus hybrid infects several species of the hyperthermophilic acidophilic crenarchaeon Sulfolobus. The open reading frame orfc68 of pSSVx encodes a 7.7 kDa protein that does not show significant sequence homology with any protein with known three-dimensional structure. EMSA (electrophoretic mobility-shift assay) experiments, DNA footprinting and CD analyses indicate that recombinant C68, purified from Escherichia coli, binds to two different operator sites that are located upstream of its own promoter. The three-dimensional structure, solved by a single-wavelength anomalous diffraction experiment on a selenomethionine derivative, shows that the protein assumes a swapped-hairpin fold, which is a distinctive fold associated with a family of prokaryotic transcription factors, such as AbrB from Bacillus subtilis. Nevertheless, C68 constitutes a novel representative of this family because it shows several peculiar structural and functional features.

Multivariate phase combination improves automated crystallographic model building

Density modification is a standard technique in macromolecular crystallography that can significantly improve an initial electron-density map. To obtain optimal results, the initial and density-modified map are combined. Current methods assume that these two maps are independent and propagate the initial map information and its accuracy indirectly through previously determined coefficients. A multivariate equation has been derived that no longer assumes independence between the initial and density-modified map, considers the observed diffraction data directly and refines the errors that can occur in a single-wavelength anomalous diffraction experiment. The equation has been implemented and tested on over 100 real data sets. The results are dramatic: the method provides significantly improved maps over the current state of the art and leads to many more structures being built automatically.

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

Structural Similarities between Thiamin-Binding Protein and Thiaminase-I Suggest a Common Ancestor,

ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 A resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor.

The application of multivariate statistical techniques improves single-wavelength anomalous diffraction phasing.

Recently, there has been a resurgence in phasing using the single-wavelength anomalous diffraction (SAD) experiment; data from a single wavelength in combination with techniques such as density modification have been used to solve macromolecular structures, even with a very small anomalous signal. Here, a formulation for SAD phasing and refinement employing multivariate statistical techniques is presented. The equation developed accounts explicitly for the correlations among the observed and calculated Friedel mates in a SAD experiment. The correlated SAD equation has been implemented and test cases performed on real diffraction data have revealed better results compared with currently used programs in terms of correlation with the final map and obtaining more reliable phase probability statistics.

Insights into peptide nucleic acid (PNA) structural features: the crystal structure of a D-lysine-based chiral PNA-DNA duplex.

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA-RNA and PNA-DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as D-Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three D-Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH2, in which pn represents a pseudopeptide link and dl represents a D-Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-A resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. The D-Lys-based chiral PNA-DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition.

A new class of gadolinium complexes employed to obtain high-phasing-power heavy-atom derivatives: results from SAD experiments with hen egg-white lysozyme and urate oxidase from Aspergillus flavus.

Seven gadolinium complexes are shown to be excellent compounds for the preparation of heavy-atom derivatives for macromolecular crystallography projects. De novo phasing has been carried out using single-wavelength anomalous diffraction (SAD) on a series of gadolinium-derivative crystals of two proteins: hen egg-white lysozyme and urate oxidase from Aspergillus flavus. Lysozyme derivative crystals were obtained by co-crystallizing the protein with the corresponding gadolinium complex at a concentration of 100 mM. Diffraction data were collected to a resolution of 1.7 A using Cu K(alpha) radiation from a rotating-anode generator, making use of the high anomalous signal of gadolinium at this wavelength. Urate oxidase derivative crystals were obtained by soaking native crystals in 100 mM gadolinium complex solutions. Diffraction data were collected to a resolution close to 3 A using X-rays at the Gd L(III) absorption edge, taking advantage of the sharp white line on that edge. For all urate oxidase derivative crystals and three of the lysozyme crystals, SAD phasing led to electron-density maps of very high quality, allowing unambiguous chain tracing. From this study, the binding effectiveness of the gadolinium complexes seems to be related to the nature of the precipitant used for crystallization. These gadolinium complexes represent a new class of high-phasing-power heavy-atom derivatives that may be used for high-throughput structure-determination projects.