Crystallization of P2 myelin protein

Single crystals of bovine P2 myelin protein have been grown in polyethylene glycol 4000 by the hanging-drop vapor diffusion method. Crystals belonging to space group P2(1)2(1)2(1) with cell dimensions a = 91.8 A, b = 99.5 A, c = 56.5 A (1 A = 0.1 nm). The diffraction pattern extends to better than 2.3 A resolution.     

Crystallization of the aspartylprotease from the human immunodeficiency virus, HIV-1

The aspartylprotease of the human immunodeficiency virus HIV-1 (NY5) has been crystallized in a form suitable for x-ray diffraction analysis. The crystals are tetragonal bipyramids and produce an x-ray diffraction pattern that exhibits the symmetry associated with space group P4(1)2(1)2 (or its enantiomorph, P4(3)2(1)2). The unit cell parameters are a = b = 50.3 A, c = 106.8 A, alpha = beta = gamma = 90 degrees; measurable diffraction intensities are observed to a resolution of 2.5 A. Density measurements indicate one molecule of 9,400 daltons/asymmetric unit. The symmetry of this space group could accommodate the proposed active dimer species of the protease if the 2-fold axis were coincident with one of the crystallographic 2-fold axes.     

Crystallization of and preliminary X-ray data for bovine carbonic anhydrase III

Crystals of bovine carbonic anhydrase III have been grown in a solution of polyethylene glycol. The crystals are monoclinic, space group P2(1), with the unit cell parameters a = 50.6 A, b = 44.7 A, c = 56.9 A, and beta = 90.3 degrees. The asymmetric unit contains 1 molecule. The diffraction pattern extends beyond 2.0-A resolution.     

Small angle x-ray scattering studies on adenovirus type 2 hexon

Adenovirus type 2 hexons have been studied in solution by small angle X-ray scattering, and the following molecular parameters determined: radius of gyration (Rg) = 4.9 nm, molecular weight (M) = 310.000, invariant volume (Vinv) = 630 mn3, maximal distance (Dmax) = 14.5--15.5 nm. A diffraction pattern was obtained up to an angular increment of h = 2.5 nm-1. Various models for the hexon have been explored by calculating the diffraction pattern from the Debye formula for 1200 spheres arranged to define the scattering volume of each model. Models were first built according to electron micrographic results. Later, preliminary results of a crystallographic study were used for model building. The experimental pattern and the pattern resulting from the model determined by crystallographic methods were compared and showed good agreement.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

Preliminary investigation of crystals of the neutral lipase from Pseudomonas fluorescens

The neutral lipase from the bacteria Pseudomonas fluorescens, marketed under the trade name LpL-200S, has been crystallized in a form suitable for X-ray diffraction analysis from 35% n-propanol at pH 8.5. The crystals are monoclinic prisms and are of space group C2 with a = 91.00 A, b = 47.17 A, c = 35.21 A and beta = 121.43 degrees. There is one molecule of the protein as the asymmetric unit of the crystals. The diffraction pattern extends to at least 1.6 A resolution and the crystals are extremely robust in terms of X-ray exposure.     

Assembly of Acanthamoeba myosin-II minifilaments. Model of anti-parallel dimers based on EM and X-ray diffraction of 2D and 3D crystals

Current models suggest that the first step in the assembly of Acanthamoeba myosin-II is anti-parallel dimerization of the coiled-coil tails with an overlap of 15 nm. Sedimentation equilibrium experiments showed that a construct containing the last 15 heptads and the non-helical tailpiece of the myosin-II tail (15T) forms dimers. To examine the structure of the 15T dimer, we grew 3D and 2D crystals suitable for X-ray diffraction and electron image analysis, respectively. For both conditions, crystals formed in related space and plane groups with similar unit cells (a=87.7 A, b=64.8 A, c=114.9 A, beta=108.0 degrees). Inspection of the X-ray diffraction pattern and molecular replacement analysis revealed the orientation of the coiled-coils in the unit cell. A 3D density map at 15A in-plane resolution derived from a tilt series of electron micrographs established the solvent content of the 3D crystals (75%, v/v), placed the coiled-coil molecules at the approximate translation in the unit cell, and revealed the symmetry relationships between molecules. On the basis of the low-resolution 3D structure, biochemical constraints, and X-ray diffraction data, we propose a model for myosin interactions in the anti-parallel dimer of coiled-coils that guide the first step of myosin-II assembly.     

X-ray diffraction by crystals of beef heart ubiquinol: cytochrome c oxidoreductase.

Beef heart mitochondrial ubiquinol:cytochrome c oxidoreductase has been crystallized in the shape of hexagonal bipyramids. At present the crystals diffract X-rays to 4.7 A. From preliminary analysis the diffraction pattern appears to be consistent with space group P6(1)22 or P6(5)22 and with unit cell parameters a = b = 212 A and c = 352 A.     

X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.     

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.     

Crystal Structure Determination of New Antimitotic Agent Bis(p-fluorobenzyl)trisulfide

The purpose of this research was to investigate the physical characteristics and crystalline structure of bis(p-fluorobenzyl)trisulfide, a new anti-tumor agent. Methods used included X-ray single crystal diffraction, X-ray powder diffraction (XRPD), Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetric (DSC) and thermogravimetric (TG) analyses. The findings obtained with X-ray single crystal diffraction showed that a monoclinic unit cell was a = 12.266(1) A, b = 4.7757(4) A, c = 25.510(1) A, beta = 104.25(1) degrees ; cell volume = 1,448.4(2) A(3), Z = 4, and space group C2/c. The XRPD studies of the four crystalline samples, obtained by recrystallization from four different solvents, indicated that they had the same diffraction patterns. The diffraction pattern stimulated from the crystal structure data is in excellent agreement with the experimental results. In addition, the identical FT-IR spectra of the four crystalline samples revealed absorption bands corresponding to S-S and C-S stretching as well as the characteristic aromatic substitution. Five percent weight loss at 163.3 degrees C was observed when TG was used to study the decomposition process in the temperature range of 20-200 degrees C. DSC also allowed for the determination of onset temperatures at 60.4(1)-60.7(3) degrees C and peak temperatures at 62.1(3)-62.4(3) degrees C for the four crystalline samples studied. The results verified that the single crystal structure shared the same crystal form with the four crystalline samples investigated.     

Crystallization and preliminary X-ray data of a phleomycin-binding protein from Streptoalloteichus hindustanus

Large single crystals of a glycopeptide resistance protein encoded by the SH-ble gene from Streptoalloteichus hindustanus have been grown using vapor diffusion techniques with ammonium sulfate as the precipitant. The diffraction pattern extends to 2.0 A resolution. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have unit cell dimensions of a = 48.8 A and c = 112.5 A.     

Crystallization of halorhodopsin from Halobacterium sp. shark

The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.     

The lyotropic liquid crystal properties of n-octyl 1-O-beta-D-glucopyranoside and related n-alkyl pyranosides

X-ray diffraction patterns have been obtained for the lyotropic phases of n-octyl 1-O-beta-D-glucopyranoside and the related n-heptyl, n-nonyl and n-decyl compounds with water. The octyl compound exhibits all three liquid crystal phases and forms a micellar solution with increasing solvation, when the crystal come into contact with water at room temperature. The X-ray diffraction patterns show a one-dimensional lamellar phase with [dx] = 28.4 A, a three-dimensional face-centered cubic phase with [a] = 51.2 A, and a two-dimensional hexagonal phase with [a] = [b] = 36.7 A. The micellar solution has a distribution pattern with a maximum at [dx] = 33.8 A. Crystals of the heptyl, nonyl and decyl derivatives form only the lamellar phases and the micellar solution on contact with water at room temperature.     

Crystallographic studies on a family B DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1.

A hyperthermostable family B DNA polymerase from the hyperthermophilic archaeon, Pyrococcus kodakaraensis strain KOD1, has been crystallized by the hanging-drop vapor diffusion method at 293 K with 2-methyl-2,4-pentanediol as the precipitant. The diffraction pattern of a crystal extends to 3.0 A resolution, and two full sets of 3.0 A resolution diffraction data for native crystals were successfully collected at 290 K and 100 K upon exposure to synchrotron radiation at KEK-PF, Japan. The crystals belong to the space group, P212121, with unit-cell dimensions of a = 112.8, b = 115.4, and c = 75.4 A at 290 K, and a = 111.9, b = 112.4, and c = 73.9 at 100 K. Structural analysis by means of the multiple isomorphous replacement method is now in progress.     

Crystallization of GST2, a human class alpha glutathione transferase

Single crystals of human GST2, a class alpha glutathione transferase have been grown in polyethylene glycol 2000 by the hanging-drop vapour diffusion method. The crystals belong to space group C2 and have cell dimensions a = 100.8 A, b = 95.4 A, c = 105.2 A and beta = 92.4 degrees. The X-ray diffraction pattern extends to better than 3 A resolution.     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

Crystal structure determination of thymoquinone by high-resolution X-ray powder diffraction

The crystal structure of 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and its thermal behavior--as necessary physical and chemical properties--were determined in order to enhance the current understanding of thymoquinone chemical action by using high resolution x-ray powder diffraction, Fourier transform infrared spectroscopy (FTIR), and 3 thermo-analytical techniques thermogravimetric analysis (TGA), differential thermal analysis (DTA), and differential scanning calorimetry (DSC). The findings obtained with high-resolution x-ray powder diffraction and molecular location methods based on a simulated annealing algorithm after Rietveld refinement showed that the triclinic unit cell was a = 6.73728(8) A, b = 6.91560(8) A, c = 10.4988(2) A, alpha = 88.864(2) degrees, beta = 82.449(1) degrees, gamma = 77.0299(9) degrees; cell volume = 472.52(1) A3, Z = 2, and space group P1. In addition, FTIR spectrum revealed absorption bands corresponding to the carbonyl and C-H stretching of aliphatic and vinylic groups characteristically observed in such p-benzoquinones. Also, a chemical decomposition process starting at 65 degrees C and ending at 213 degrees C was noted when TGA was used. DSC allowed for the determination of onset at 43.55 degrees C and a melting enthalpy value of DeltaH(m) = 110.6 J/g. The low value obtained for the fusion point displayed a van der Waals pattern for molecular binding, and the thermograms performed evidence that thymoquinone can only be found in crystalline triclinic form, as determined by DRX methods.     

Crystallization and preliminary crystallographic data of a Streptomyces scabies extracellular esterase.

X-ray quality single crystals of an extracellular esterase from pathogenic Streptomyces scabies were obtained by the hanging drop method. The crystals are monoclinic (space group C2, a = 161.1 A, b = 51.2 A, c = 124.2 A, beta = 100.6 degrees) with two molecules related by a noncrystallographic dyad in the asymmetric unit, with a solvent content of approximately 64%. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube CuK alpha radiation. The study has been initiated in order to elucidate the mechanism of this unusual non-serine-dependent esterase, and to gain better understanding of the molecular basis of the pathogenesis of the scab disease.     

Characterization of crystals of an Fab fragment of a murine monoclonal antibody.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.