Rotating-anode X-ray Generator Research Articles

Crystallization and preliminary X-ray studies of cold-active protein-tyrosine phosphatase of Shewanella sp.

Cold-active protein-tyrosine phosphatase (PTPase) of Shewanella sp. was expressed, purified and crystallized using the hanging-drop vapour-diffusion method at two different pH values (4.6 and 8.5). Both crystals are orthorhombic and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.4, b = 76.8, c = 81.0 A (pH 8.5) and a = 57.1, b = 77.0 and c = 81.5 A (pH 4.6). Diffraction data to 1.82 A for the pH 8.5 crystal and 2.33 A for the pH 4.6 crystal were collected on a multiwire area detector using a rotating-anode X-ray generator.

Isolation, purification and preliminary X-ray characterization of Cpn60-2 (65 kDa heat-shock protein) from Mycobacterium tuberculosis.

Cpn60-2 is a member of a unique family of putative molecular chaperones homologous to GroEL (Cpn60) but of unknown function and found only in Mycobacterium tuberculosis and closely related species. Cpn60-2 has mainly been studied for its strong immunogenity. Here, the purification, crystallization and preliminary crystallographic analysis of M. tuberculosis Cpn60-2 are reported. The crystals belong to space group P2, with unit-cell parameters a = 57, b = 115.5, c = 81.5 A, beta = 95.5 degrees, and contain a dimer in the asymmetric unit. The crystals diffract to 4.0 A using a Cu rotating-anode X-ray generator.

X-ray characterization of crystal perfection and surface contamination in large-diameter silicon wafers

This paper reviews recent development of X-ray techniques, which cover laboratory experiments and synchrotron radiation (SR) applications, to detect crystal imperfections and surface contamination in large-diameter Czochralski-grown silicon (CZ-Si) wafers. While process-induced defects can be easily detected by a large-sized Lang camera, it is difficult to observe grown-in microdefects and slight impurity-inhomogeneity even when the double-crystal method is applied. SR plane-wave X-ray topography has overcome this difficulty, except for observing void defects, with the help of its high strain sensitivity although it cannot be directly applied to large Si wafers because of their warpage. Recently, SR X-ray topography using a 300-mm-wide monochromatic beam has been employed for measuring the warpage of 200- and 300-mm wafers as well as inspecting surface damage caused by various steps of wafer-manufacturing. Although energy-dispersive total-reflection X-ray fluorescence analysis using a rotating-anode X-ray generator is now widely used for detecting traces of metallic impurities on the surface of 300-mm Si wafers, the requirement of a large scale integrated circuit miniaturization has promoted the combination of a vapor-phase decomposition technique and the TXRF for lowering the lower-limit of detection (LLD). SR-TXRF using an ED solid-state detector is effective in nondestructive and low LLD features, while newly developed wavelength-dispersive (WD) SR-TXRF in good energy resolution. The combined use of the laboratory and SR experiments leads to precise information about crystal perfection and surface contamination in large-diameter Si wafers.

The Crystal Structure of the Endothelial Protein C Receptor and a Bound Phospholipid

The endothelial cell protein C receptor (EPCR) shares approximately 20% sequence identity with the major histocompatibility complex class 1/CD1 family of molecules, accelerates the thrombin-thrombomodulin-dependent generation of activated protein C, a natural anticoagulant, binds to activated neutrophils, and can undergo translocation from the plasma membrane to the nucleus. Blocking protein C/activated protein C binding to the receptor inhibits not only protein C activation but the ability of the host to respond appropriately to bacterial challenge, exacerbating both the coagulant and inflammatory responses. To understand how EPCR accomplishes these multiple tasks, we solved the crystal structure of EPCR alone and in complex with the phospholipid binding domain of protein C. The structures were strikingly similar to CD1d. A tightly bound phospholipid resides in the groove typically involved in antigen presentation. The protein C binding site is outside this conserved groove and is distal from the membrane-spanning domain. Extraction of the lipid resulted in loss of protein C binding, which could be restored by lipid reconstitution. CD1d augments the immune response by presenting glycolipid antigens. The EPCR structure is a model for how CD1d binds lipids and further suggests additional potential functions for EPCR in immune regulation, possibly including the anti-phospholipid syndrome.

Crystallographic study of a naturally occurring trans-splicing intein from Synechocystis sp. PCC 6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been purified and crystallized using PEG 8K as precipitant. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 58.5, c = 70.2 A. It has one molecule per asymmetric unit and diffracts to beyond 2.9 A under cryoconditions (110 K) using a Cu rotating-anode X-ray generator in-house.

Crystallization and preliminary X-ray analysis of a Trx domain of human thioredoxin-like protein.

The Trx domain of human thioredoxin-like protein has been purified and crystallized using ammonium sulfate as precipitant. The crystal belongs to space group C2, with unit-cell parameters a = 87.5, b = 48.5, c = 29.8 A, beta = 99.59 degrees. It has one molecule per asymmetric unit and diffracts beyond 2.2 A under cryoconditions (100 K) using an in-house Cu rotating-anode X-ray generator.

Applications of anomalous scattering from S atoms for improved phasing of protein diffraction data collected at Cu Kalpha wavelength.

The anomalous signal of S atoms is easily detected at the Cu Kalpha wavelength of a non-synchrotron source with current data-collection methods. The position of sulfur and other anomalous scatterers can be located through an anomalous difference Fourier map (F+ - F-, phi(calc) - 90 degrees). It has been discovered experimentally that even low-quality preliminary phases are often sufficient to find anomalous scatterers. Their anomalous signal in the native crystal can contribute to significant improvement in phase refinement. This technique has been applied to solve the crystal structures of orthorhombic lysozyme and thaumatin. Furthermore, the structure of trypsin was solved using only the diffraction data set from a native crystal collected at a single wavelength (Cu Kalpha) from a rotating-anode X-ray generator. The anomalous scattering of sulfur was essential to solve the structure of trypsin which was initially phased from a single intrinsic Ca2+ atom. The positions of the S atoms of lysozyme and thaumatin were found using the initial SIRAS phases and used in phase refinement. The overall figures of merit and those in each resolution shell were consistently improved. This resulted in much improved electron-density maps even when the diffraction data were limited to 2.5 A resolution or worse. Furthermore, peaks from S atoms and other anomalous scatterers in anomalous difference Fourier maps can confirm the tracing of the peptide chain and also provide independent unbiased confirmation of molecular-replacement results. Thus, the anomalous signal of S atoms can contribute to many aspects of solving protein structures and should be used routinely.

Characterization and crystallization of a novel Sarcocystis muris lectin, SML-2.

A novel lectin (SML-2) consisting of 138 amino acids was isolated from cyst merozoites of Sarcocystis muris and sequenced by Edman degradation and mass spectrometry. All 12 cysteinyl residues are involved in disulfide bridges, four of which are attributed to a characteristic pattern of cysteines as found in the so-called PAN-module superfamily. Crystals of SML-2 diffracting to 2.1 A resolution at a synchrotron were grown by the hanging-drop vapour-diffusion technique. They belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 53.6, b = 128.8, c = 158.2 A and eight molecules in the asymmetric unit. SML-2 cocrystallized with Au galactose results in two different crystal forms. The first form is isomorphous with the native crystals and the second form adopts space group C222(1), with unit-cell parameters a = 74.7, b = 82.0, c = 131.0 A, and diffracts to 2.4 A at a rotating-anode X-ray generator.

The Role of Development of the Rotating Anode X-ray Generator and the Use of Imaging Plates in Powder Diffractometer Instrumentation

The rotating-anode X-ray generator has played a very important role in the instrumentation of X-ray diffraction (XRD), which is a necessary tool in materials science. Such a rotating-anode X-ray generator was developed in 1952 in response to the demands of the community of powder diffraction crystallographers in Japan, whose long history extends to 1912. The rotating-anode generator has been improved with the advance of new technologies and over time has become more reliable and more powerful. This will be true also for the future. At present, an efficient parallel X-ray beam is obtained by combining the rotating-anode X-ray generator with newly developed X-ray optics based on a synthetic multilayer optic. Several applications to the characterization of advanced materials and thin film by XRD are presented. The significance of the use of imaging plate systems, two-dimensional digital detectors, in the X-ray characterization of materials is also given.

The Annual James L. Waters Symposium at Pittcon (2000)

The topic of the eleventh Waters Symposium, held in March 2000, was X-ray diffraction of powders and thin films, and three of the papers are featured in this issue of the Journal. In the introductory paper, Ron Jenkins (International Centre for Diffraction Data) presents an overview of the development of powder diffraction instrumentation. This is followed by a description of the development of the rotating anode X-ray generator and of imaging plates by Jimpei Harada (Rigaku Corporation). Finally, in the third paper, Tom Ryan (Philips Analytical) discusses the special requirements for instrumentation suitable for diffraction of thin films such as those that are important in semiconductors, recording media, and other technologies.

In-house Anomalous X-ray Scattering Analysis for the Amorphous Zr<SUB>60</SUB>Al<SUB>15</SUB>Ni<SUB>25</SUB> Alloy

In-house anomalous X-ray scattering (AXS) analysis coupled with white radiation produced from a rotating anode X-ray generator and a solid state detector has been tested by obtaining the environmental structure around Zr in the amorphous Zr 60 Al 15 Ni 25 alloy. The AXS measurement with the conventional X-ray source was successfully carried out with a newly constructed system by enhancing the intensity of incident X-rays. The present AXS data for Zr in the amorphous Zr 60 Al 15 Ni 25 alloy was found to agree well with the previous results obtained by monochromatic X-rays produced from a synchrotron radiation source.

Crystallization and preliminary X-ray crystallographic analysis of DFPase from Loligo vulgaris.

'Squid-type' diisopropylfluorophosphatases (DFPases), a subclass of the phosphotriesterases, are enzymes capable of hydrolysing organophosphorus nerve agents. To date, no three-dimensional structure of a 'squid-type' DFPase is known. Here, the crystallization of the DFPase originally isolated from head ganglion of the squid Loligo vulgaris is reported. The protein has been heterologously expressed in Escherichia coli, purified to homogeneity and subsequently crystallized. The protein crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.1, b = 82.1, c = 86.6 A and one monomer per asymmetric unit. Under cryoconditions (120 K) the crystals diffracted beyond 2.0 A using a Cu rotating-anode X-ray generator.

Expression, purification, crystallization and preliminary X-ray analysis of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase.

A recombinant form of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (E.C. 3.2.2.9) has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique. While several different crystallization conditions were obtained, only one set of conditions yielded crystals suitable for X-ray diffraction analysis. These crystals grow as diamond-shaped wedges, with unit-cell parameters a = 50.92, b = 133.99, c = 70.88 A, alpha = beta = gamma = 90 degrees. The crystals belong to space group P2(1)2(1)2 and diffract to a minimum d spacing of 2.3 A on a MAR345 image plate with a Rigaku RU-200 rotating-anode X-ray generator. On the basis of density calculations, two monomers are predicted per asymmetric unit (Matthews coefficient, V(M) = 2.37 A(3) Da(-1)), with a solvent content of 48%.

Direct observation of in-plane texture in cobalt recording media by means of a laboratory-scale x-ray diffractometer

A new diffractometer was developed for measuring in-plane diffraction by employing an advanced parabolic multilayer and a rotating anode x-ray generator. By means of the diffractometer, the in-plane Co(002) diffraction peak and its lateral anisotropy was successfully observed in cobalt thin films of magnetic recording media, which had so far been difficult to observe with a conventional θ/2θ diffractometer. In-plane diffraction is useful for analyzing such a lateral structure of thin films. It was possible to estimate lattice constants, crystallite size and the texture of the crystallites of cobalt thin films from the observed in-plane diffraction data. It was found that the magnetic properties of these disks had a close relationship with the crystallographic structure of cobalt films obtained by the present in-plane diffraction. Copyright © 1999 John Wiley & Sons, Ltd.

Crystallization of L-aspartate oxidase, the first enzyme in the bacterial de novo biosynthesis of NAD.

The flavoenzyme L-aspartate oxidase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The crystals belong to space group P3121 (or P3221) with unit-cell parameters a = b = 84.9, c = 159.9 A. A solvent content of 42% corresponds to a monomer (60 kDa) in the asymmetric unit. A complete 2.8 A resolution data set was collected using a rotating-anode X-ray generator.

Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase.

Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs specific for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase regulator associated with focal-adhesion kinase (GRAF). It shares approximately 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs, but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator.

Structures of monoclinic lysozyme iodide at 1.6 A and of triclinic lysozyme nitrate at 1.1 A.

Hen egg-white lysozyme is one of the most thoroughly studied of enzymes and has been the subject of study by many methods, including X-ray crystallography. The present work extends the X-ray crystallography to higher resolution, includes the positions of the anions, and examines the contacts of the neighbors in greater detail. Data were collected at room temperature on a Rigaku R-axis area detector with rotating-anode X-ray generator to 1.6 A resolution for monoclinic lysozyme iodide at pH 4.0, to 1.8 A for monoclinic lysozyme iodide at pH 8.0, and to 1.1 A resolution for triclinic lysozyme nitrate at pH 4.5. The structures have been refined by SHELX93 with the expected number of anion sites being accounted for. Two regions of the protein have been found to be variable: residues 65-75 and 99-104. Except for 65-75 and 99-104, lysozyme is a very stable molecule with the crystal forms being held together by the electrostatic contacts of the anions and by layers of water molecules. The anion positions can be described as paired half sites, each half being contributed by a different lysozyme molecule. The many different crystal forms of lysozyme may be due to different combinations of the many such half sites on the surface. A hypothesis is presented for lysozyme in the different crystal forms and which may be extended to behavior in solution. Suggestions for future crystallographic research are proposed, involving anions of different shape and charge.

Construction of a Two-Dimensional Ultra-Small-Angle X-ray Scattering Apparatus

A two-dimensional ultra-small-angle X-ray scattering (USAXS) apparatus was constructed using a rotating-anode X-ray generator and a Bonse–Hart camera. In this camera, two sets of two channel-cut single crystals were used to collimate the X-ray beam in both the horizontal and the vertical planes. The measured intensity profile of the direct beam showed a high small-angle resolution in all directions on the detector plane. The full width at half-maximum was 17′′, indicating that the apparatus can be applied to structural analysis in the range up to 2 μm, even for directionally oriented samples. One- and two-dimensional USAXS profiles from colloidal silica powder agreed well with each other, showing that the desmearing procedure adopted in the previous one-dimensional USAXS experiments were justified.

Pressurized ion chamber for low energy radiation monitoring

Ion chambers that can respond to gamma energies as low as 35 keV have been developed for environmental and area monitoring for low energy X-ray background at the rotating anode X-ray generator facility and other accelerators at CAT, Indore. They are expected to measure exposure levels ranging from 10 μr/h to 100 mr/h. These chambers have an all-welded aluminium construction and pressurized nitrogen filling. Explosion welded SS-Al clad material has been used for the welding of SS-to-alumina insulators. Out of the two ion chambers developed, the environmental ion chamber has a 25 1 sensitive volume filled at 85 psi and detects radiation exposure levels from 10μr/h to 10 mr/h. The 800 cc area monitor ion chamber filled at 120 psi can be used from 10 mr/h to 10 r/h with a portable survey instrument. These chambers have an energy response between 35 keV and 1.25 MeV that is uniform within ± 9%.

Laboratory Stopped-Flow XAFS Apparatus. Structure Determination of the Short-Lived Peroxochromium Intermediate Formed during Reduction Process of Chromate(VI) Ion by Hydrogen Peroxide

In order to determine the structure parameters of the reaction intermediate formed during the reduction process of chromate(VI) ion by hydrogen peroxide, a laboratory stopped-flow X-ray absorption fine structure (XAFS ) apparatus, which was constructed by a rotating anode X-ray generator, an energy scanning spectrometer, a stopped-flow assembly, and X-ray detectors, was newly developed. Using the apparatus, the Cr K -edge XAFS spectrum of the reaction intermediate was measured. One oxo group, two peroxo groups, and one water molecule are coordinated to the Cr(VI) center in the intermediate, CrO(O2)2(H2O ) , with Cr - O bond lengths of 157, 168, and 193 pm, respectively. The geometry around the Cr(VI) center is probably 6-coordinate pentagonal pyramidal.