Optics Concept for a Pair of Undulator Beamlines for MX.

We describe a concept for x-ray optics to feed a pair of macromolecular crystallography (MX) beamlines which view canted undulator radiation sources in the same storage ring straight section. It can be deployed at NSLS-II and at other low-emittance third-generation synchrotron radiation sources where canted undulators are permitted, and makes the most of these sources and beamline floor space, even when the horizontal angle between the two canted undulator emissions is as little as 1-2 mrad. The concept adopts the beam-separation principles employed at the 23-ID (GM/CA-CAT) beamlines at the Advanced Photon Source (APS), wherein tandem horizontally-deflecting mirrors separate one undulator beam from the other, following monochromatization by a double-crystal monochromator. The scheme described here would, in contrast, deliver the two tunable monochromatic undulator beams to separate endstations that address rather different and somewhat complementary purposes, with further beam conditioning imposed as required. A downstream microfocusing beamline would employ dual-stage focusing for work at the micron scale and, unique to this design, switch to single stage focusing for larger beams. On the other hand, the upstream, more highly automated beamline would only employ single stage focusing.

Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II.

Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a beta-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4(1) with unit-cell parameters a = b = 150.17, c = 77.41 A were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 A, beta = 113.6 degrees. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high thermal and pH stabilities, which are required for a number of biotechnological and industrial applications.

Application of a neural network in high-throughput protein crystallography.

High-throughput protein crystallography requires the automation of multiple steps used in the protein structure determination. One crucial step is to find and monitor the crystal quality on the basis of its diffraction pattern. It is often time-consuming to scan protein crystals when selecting a good candidate for exposure. The use of neural networks for this purpose is explored. A dynamic neural network algorithm to achieve a fast convergence and high-speed image recognition has been developed. On the test set a 96% success rate in identifying properly the quality of the crystal has been achieved.

Formation of a tyrosyl radical intermediate in Proteus mirabilis catalase by directed mutagenesis and consequences for nucleotide reactivity.

Proteus mirabilis catalase (PMC) belongs to the family of NADPH binding catalases. The function of NADPH in these enzymes is still a matter of debate. This study presents the effects of two independent phenylalanine mutations (F194 and F215), located between NADPH and heme in the PMC structure. The phenylalanines were replaced with tyrosines which we predicted could carry radicals in a NADPH-heme electron transfer. The X-ray crystal structures of the two mutants indicated that neither the binding site of NADPH nor the immediate environment of the residues was affected by the mutations. Measurements using H2O2 as a substrate confirmed that the variants were as active as the native enzyme. With equivalent amounts of peroxoacetic acid, wild-type PMC, F215Y PMC, and beef liver catalase (BLC) formed a stable compound I, while the F194Y PMC variant produced a compound I which was rapidly transformed into compound II and a tyrosyl radical. EPR studies showed that this radical, generated by the oxidation of Y194, was not related to the previously observed radical in BLC, located on Y369. In the presence of excess NADPH, compound I was reduced to a resting enzyme (k(obs) = 1.7 min(-1)) in a two-electron process. This was independent of the enzyme's origin and did not require any thus far identified tyrosyl radicals. Conversely, the presence of a tyrosyl radical in F194Y PMC greatly enhanced the oxidation of reduced beta-nicotinamide mononucleotide under a steady-state H2O2 flow with observable compound II. This process could involve a one-electron reduction of compound I via Y194.

Synchrotron X-ray reciprocal-space mapping, topography and diffraction resolution studies of macromolecular crystal quality.

A comprehensive study of microgravity and ground-grown chicken egg-white lysozyme crystals is presented using synchrotron X-ray reciprocal-space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed reduced intrinsic mosaicities on average, but no differences in terms of strain over their ground-grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the ground-control case only a small volume of the crystal contributed to the intensity at the diffraction peak. The techniques prove to be highly complementary, with the reciprocal-space mapping providing a quantitative measure of the crystal mosaicity and strain (or variation in lattice spacing) and the topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out at the synchrotron.

X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli.

Type 1 pili-adhesive fibers expressed in most members of the Enterobacteriaceae family-mediate binding to mannose receptors on host cells through the FimH adhesin. Pilus biogenesis proceeds by way of the chaperone/usher pathway. The x-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli at 2.5 angstrom resolution reveals the basis for carbohydrate recognition and for pilus assembly. The carboxyl-terminal pilin domain of FimH has an immunoglobulin-like fold, except that the seventh strand is missing, leaving part of the hydrophobic core exposed. A donor strand complementation mechanism in which the chaperone donates a strand to complete the pilin domain explains the basis for both chaperone function and pilus biogenesis.

X-ray topography of tetragonal lysozyme grown by the temperature-controlled technique.

Growth-induced defects in lysozyme crystals were observed by white-beam and monochromatic X-ray topography at the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory (BNL). The topographic methods were non-destructive to the extent that traditional diffraction data collection could be performed to high resolution after topography. It was found that changes in growth parameters, defect concentration as detected by X-ray topography, and the diffraction quality obtainable from the crystals were all strongly correlated. In addition, crystals with fewer defects showed lower mosaicity and higher diffraction resolution as expected.

Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6.

The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43,808 and pI of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme. The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive. The crystals belong to a primitive trigonal crystal system (space group P3(1) or P3(2)) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A. These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution. The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays. A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions.

Expression, purification and characterization of recombinant crambin.

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues), has been successfully expressed in Escherichia coli from an artificial, synthetic gene. Several expression systems were investigated. Ultimately, crambin was successfully expressed as a fusion protein with the maltose binding protein, which was purified by affinity chromatography. Crambin expressed as a C-terminal domain was then cleaved from the fusion protein with Factor Xa protease and purified. Circular dichroism spectroscopy and amino acid analysis suggested that the purified material was identical to crambin isolated from seed. For positive identification the protein was crystallized from an ethanol-water solution, by a novel method involving the inclusion of phospholipids in the crystallization buffer, and then subjected to crystallographic analysis. Diffraction data were collected at the Brookhaven synchrotron (beamline-X12C) to a resolution of 1.32 A at 150 K. The structure, refined to an R value of 9.6%, confirmed that the cloned protein was crambin. The availability of cloned crambin will allow site-specific mutagenesis studies to be performed on the protein known to the highest resolution.

Repartitioning of NaCl and protein impurities in lysozyme crystallization.

Nonuniform precipitant and impurity incorporation in protein crystals can cause lattice strain and, thus, possibly decrease the X-ray diffraction resolution. To address this issue, a series of crystallization experiments were carried out, in which initial supersaturation, NaCl concentration, protein purity level and crystallized fraction were varied. Lysozyme and protein impurities, as well as sodium and chloride were independently determined in the initial solution, supernatant and crystals. The segregation coefficients for Na(+) and Cl(-) were found to be independent of supersaturation and NaCl concentration, and decreased with crystallized fraction/crystal size. Numerical evaluation of the extensive body of data, based on a nucleation-growth-repartitioning model, suggests a core of approximately 40 micro m in which salt is incorporated in much greater concentrations than during later growth. Small crystals containing higher amounts of incorporated NaCl also had higher protein impurity contents. This suggests that the excess salt is associated with the protein impurities in the core. X-ray topography revealed strain fields in the center of the crystals comparable in size to the inferred core. The growth rates of crystals smaller than 30-40 micro m in size were consistently 1.5-2 times lower than those of larger crystals, presumably due to higher chemical potentials in the core.

Crystallization and preliminary X-ray investigation of lipoxygenase-3 from soybeans.

Soybean lipoxygenase-3 has been crystallized by the vapor diffusion method in 16-20% polyethylene glycol (average M(r), 3,400), 0.2 M sodium acetate buffer, pH 5.7, at 21 degrees C, at a protein concentration of 8-15 mg/mL. The crystals, which diffract to 3-A spacings, belong to the monoclinic space group C2. Cell constants are a = 111.9, b = 136.4, and c = 61.6 A and beta = 95.7 degrees. The calculated value of Matthews's constant, Vm = 2.48 A3/kDa, is consistent with the presence of one molecule of lipoxygenase per crystallographic asymmetric unit (Z = 4).