Crystallization and preliminary X-ray diffraction results of snowdrop (Galanthus nivalis) lectin

Well-ordered single crystals have been grown for a mannose-specific lectin from snowdrop (Galanthus nivalis) bulbs in the presence of methyl-alpha-D-mannoside. The space group symmetry is consistent with the orthorhombic space group P2(1)2(1)2(1). The unit cell parameters are a = 140.0 A, b = 64.7 A, c = 62.0 A. The asymmetric unit can accommodate a tetramer. The functional molecule (50,000 daltons) consists of four identical subunits and is highly specific for alpha 1,3-linked mannose oligosaccharides.     

Preliminary crystallographic study of the phenylalanyl-tRNA synthetase from Thermus thermophilus HB8

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) from the extreme thermophile Thermus thermophilus HB8 has been isolated and crystallized. The enzyme was found to consist of two types of subunits with molecular masses 38 X 10(3) (alpha) and 94 X 10(3) (beta) and is likely to be a tetrameric protein with a molecular mass of about 260 X 10(3) (alpha 2 beta 2). Crystals of phenylalanyl-tRNA synthetase were grown by the hanging-drop technique at 4 degrees C in the presence of ammonium sulfate. Trigonal crystals, space group P3(1)21, with cell dimensions a = b = 176 A and c = 142 A (1 A = 0.1 nm), are suitable for medium-resolution X-ray analysis.     

Preliminary crystallographic data and quaternary structural implications of the central subunit of the multi-subunit complex transcarboxylase

The hexameric central subunit (Mr = 360,000) of the multi-subunit complex transcarboxylase has been crystallized by bulk dialysis against 250 mM-sodium acetate (pH 5.5). The crystals are cubic, a = 193.1 A, space group P4(1)32 or enantiomorph. The number of molecules per unit cell is four and was deduced from the density of the crystals (1.10 g cm-3) and the mother liquor (1.01 g cm-3) and the specific volume of the protein calculated from molecular dimensions obtained from electron microscopy studies. Four molecules per cell requires the central subunits to lie on 3-fold axes, which are perpendicular to 2-fold rotation axes, so that the molecules satisfy 32 symmetry giving one subunit as the asymmetric unit. Of the four possible models that have been considered for the quaternary structure of transcarboxylase, only that with antiparallel subunits, two sets of isologous binding sites and D3 symmetry is in agreement with the symmetry requirements of the cubic crystals.     

Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1

Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.     

Electron crystallographic study of photosystem II of the cyanobacterium Synechococcus elongatus

The determination of the structure of PSII at high resolution is required in order to fully understand its reaction mechanisms. Two-dimensional crystals of purified highly active Synechococcus elongatus PSII dimers were obtained by in vitro reconstitution. Images of these crystals were recorded by electron cryo-microscopy, and their analysis revealed they belong to the two-sided plane group p22(1)2(1), with unit cell parameters a = 121 A, b = 333 A, and alpha = 90 degrees. From these crystals, a projection map was calculated to a resolution of approximately 16 A. The reliability of this projection map is confirmed by its close agreement with the recently presented three-dimensional model of the same complex obtained by X-ray crystallography. Comparison of the projection map of the Synechococcus elongatus PSII complex with data obtained by electron crystallography of the spinach PSII core dimer reveals a similar organization of the main transmembrane subunits. However, some differences in density distribution between the cyanobacterial and higher plant PSII complexes exist, especially in the outer region of the complex between CP43 and cytochrome b(559) and adjacent to the B-helix of the D1 protein. These differences are discussed in terms of the number and organization of some of the PSII low molecular weight subunits.     

Crystallization and preliminary x-ray investigation of purine-nucleoside phosphorylase from Escherichia coli

Crystals of purine-nucleoside phosphorylase from Escherichia coli have been grown from solutions of ammonium sulfate. The crystals are hexagonal with space group P6(1)22 or P6(5)22; the axes are alpha = 106.5 A and c = 241.3 A. The crystals are moderately stable to x-rays and diffract beyond 3.0-A resolution. It appears that the molecule, which is a hexamer, utilizes the 2-fold symmetry of the space group, resulting in three subunits/asymmetric unit.     

Crystallization and preliminary crystallographic study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

3 alpha, 20 beta-Hydroxysteroid dehydrogenase, an NADH-dependent oxidoreductase isolated from Streptomyces hydrogenans , is a tetramer containing four subunits each of Mr 25,000. The enzyme has been crystallized by the vapor diffusion technique using either phosphate or borate buffered ammonium sulfate (pH between 6.0 and 8.7) as the precipitant. The crystals are hexagonal bipyramids ; they have the symmetry of space group P6(4)22 (or P6(2)22), with unit cell dimensions a = 127.3 A, c = 112.2 A. Volume and density considerations imply that the crystallographic asymmetric unit contains two monomers, and therefore that the tetramer possesses a 2-fold axis of symmetry that is coincident with a crystallographic 2-fold symmetry element.     

Crystallization and preliminary X-ray crystallographic data of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium

The tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium can be crystallized by the method of vapor diffusion from solutions of polyethylene glycol 8000 and various salts. Thin needles are obtained in the presence of a monovalent cation (Na+), thicker crystals are obtained in the presence of divalent cations (Mg2+, Mn2+, Fe2+, Ca2+, Zn2+), and square-faced crystals are obtained in the presence of spermine. Although the spermine and Mg2+ crystals differ in morphology, they are both monoclinic and in space group C2 with a = 184.5 A, b = 62.4 A, c = 67.7 A, beta = 94 degrees 40', and one alpha beta pair of Mr = 71,700/asymmetric unit. The crystals appear reasonably resistant to radiation damage and should be suitable for a complete structure investigation. The separated S. typhimurium alpha, holo-beta 2, and apo-beta 2 subunits do not crystallize under these conditions nor do the alpha 2 beta 2 complex or the alpha- or holo-beta 2 subunits from Escherichia coli or from an interspecies hybrid.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Crystallization of and preliminary X-ray data for the mouse major urinary protein and rat alpha-2u globulin.

Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution.     

Two-dimensional crystallization of avidin on biotinylated lipid monolayers.

Two-dimensional crystals of avidin were obtained on mixed lipid monolayers containing biotinylated lipids (N-biotinyl-dipalmitoyl-L-alpha-phosphatidyl ethanolamine and dioleoyl phosphatidyl choline) by specific interaction. Image analysis of electron micrographs of these crystals revealed p2 symmetry with the unit cell parameters a = 66 +/- 2 A, b = 68 +/- 1 A, and gamma = 121 +/- 4 degrees. The projection map showed, at a resolution of about 27 A, that the four subunits within one avidin molecule are separated into two parts. Comparison between avidin and streptavidin reveals that avidin molecule binds to the lipid monolayer in an orientation similar to that of streptavidin.     

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.     

Crystallization and preliminary crystallographic investigation of methanol dehydrogenase from Methylobacterium extorquens AM1.

Single crystals of methanol dehydrogenase (MDH) from Methylobacterium extorquens AM1 have been grown by the vapour diffusion method. These crystals diffract to beyond 2 A resolution and are suitable for X-ray crystallography. They belong to the orthorhombic space group P2(1)2(1)2(1) and have the following unit cell parameters: a = 66.79 A, b = 108.9 A, c = 188.9 A. One asymmetric unit contains an alpha 2 beta 2 tetramer of MDH and the location of the non-crystallographic 2-fold symmetry axis of this tetramer is defined by the paired positions of the binding sites of heavy atoms in four MDH-derivatives.     

Proteins of the Bacillus stearothermophilus ribosome. Crystallization of proteins L30 and S5

Proteins L30 and S5 from the 50 S and 30 S subunits, respectively, of the Bacillus stearothermophilus ribosome have been crystallized. L30 crystals are tetragonal and the space group is P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 46.3 A and c = 61.4 A. S5 crystals are trigonal with the space group P3(1)21 (or P3(2)21) and cell dimensions a = b = 59.3 A and c = 109.8 A. In both cases, there appears to be a single molecule in the asymmetric unit.     

Crystallization and preliminary X-ray analysis of methylamine-treated alpha 2-macroglobulin and 3 alpha 2-macroglobulin-proteinase complexes.

Crystals of methylamine-treated alpha 2-macroglobulin (alpha 2M-MA), alpha 2-macroglobulin in complex with two molecules of trypsin, alpha 2M-T2, one molecule of plasmin, alpha 2M-PL, and one molecule of plasmin followed by methylamine-treatment, alpha 2M-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of alpha 2M-MA diffracted to at least 9 A resolution, crystals of alpha 2M-T2 diffracted to 10 A resolution and crystals of alpha 2M-PL and alpha 2M-PL(MA) diffracted to 11 A resolution. For alpha 2M-MA the cell parameters were determined as: a=b=257 A, c=555 A; and for alpha 2M-T2 as: a=b=247 A, c=559 A. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of alpha 2M-MA and alpha 2M-T2 contain one 360 kDa alpha 2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, Vm, was estimated at 5.6 A3/Da. The crystal parameters of alpha 2M-PL and alpha 2M-PL(MA) were not determined.     

Crystallization and preliminary X-ray study of AMP nucleosidase

Adenosine-5'-monophosphate nucleosidase from Escherichia coli has been crystallized in the presence of its strong competitive inhibitor formycin 5'-monophosphate and its allosteric activator adenosine 5'-triphosphate. Crystals are tetragonal bipyramids which grow to 1.2 mm in the longest dimension, are resistant to radiation damage, and diffract to a resolution of 3.5 A. The space group is P4(1)2(1)2 or P4(3)2(1)2, and the unit cell dimensions are a = 120.1 A and c = 243.7 A. The asymmetric unit is estimated to contain four subunits of 52,000 daltons. The crystals appear suitable for single crystal x-ray structure investigation.     

Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 A

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.     

Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Crystallization and preliminary X-ray crystallographic analysis of Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

S-adenosyl-l-homocysteine hydrolase from a malaria parasite Plasmodium falciparum (PfSAHH) has been crystallized by the vapor diffusion method. The crystals belong to an orthorhombic space group P212121 with the cell dimensions of a = 76.66 A, b = 86.31 A, and c = 335.6 A. There are four subunits (one tetramer) per asymmetric unit. X-ray diffraction data have been collected up to 2.8 A resolution. Self-rotation function studies suggest that the tetrameric PfSAHH molecule has the 222 point group symmetry.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.