Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.     

Two crystal forms of the lentil lectin diffract to high resolution.

The legume lectins are an important class of polysaccharide-binding proteins with a wide range of biochemical and immunological applications. Two high-resolution crystal forms are obtained for the lentil (Lens culinaris) lectin: a monoclinic P21 and an orthorhombic P212121. The unit cell dimensions for the monoclinic form are a = 58.0 A, b = 56.0 A, c = 82.1 A, beta = 104.4 degrees, while for the orthorhombic form a = 56.4 A, b = 74.6 A, c = 124.9 A. The asymmetric unit contains one dimer in both cases. The crystals diffract to 1.7 A resolution using synchrotron radiation. Preliminary data have been collected to 2.3 A on both crystal forms using a conventional X-ray source.     

Cinnamomin: separation,crystallization and preliminary X-ray diffraction study

Cinnamomin from Cinnamonum camphora seeds, a type II ribosome-inactivating protein that interferes with protein biosynthesis in mammalian cells, can induce the apoptosis of carcinoma cells and be used as an insecticide. A rapid and improved method has been developed for the extraction and purification of cinnamomin from camphora seed. Purification of cinnamomin is achieved with two successive steps of hydrophobic interaction chromatography carried out on a fast protein liquid chromatography (FPLC) system. Crystals suitable for X-ray diffraction analysis were obtained by vapor diffusion method. A complete data set at 2.8 A resolution has been collected. Data indexation and refinement indicate that the crystal is orthorhombic with space group P2(1)2(1)2(1) and unit cell dimensions a = 52.39 A, b = 126.33 A, c = 161.45 A. There are two molecules per asymmetric unit. Initial phasing by molecular replacement method yielded a solution, which will contribute to the structure determination. A molecular model will further the understanding of the mechanism of cinnamomin function. The latter will be combined with bio-informatics to facilitate the medical and other applications of cinnamomin.     

Crystallization and preliminary X-ray study of pig lens aldose reductase

Crystals of pig lens aldose reductase have been grown from polyethylene glycol solutions at pH 6.2 and analysed by X-ray diffraction. Two crystal forms were obtained. The first belongs to space group P1 with unit cell dimensions a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees, gamma = 79.0 degrees, with four molecules in the unit cell related by a 222 non-crystallographic symmetry. The second crystal form is hexagonal. The space group is P6(2)22 with a = b = 101 A, c = 257 A and two molecules in the asymmetric unit. Both forms are suitable for X-ray structure analysis to better than 3 A resolution.     

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.     

Crystallization and preliminary diffraction analysis of cholesterol esterase from Candida cylindracea

Cholesterol esterase (EC 3.1.1.13) from the microorganism Candida cylindracea has been crystallized in two forms. Crystals, typically 0.30 x 0.15 x 0.10 mm in size, diffract rotating anode generated x-rays to beyond 3 A are suitable for data collection for an x-ray crystallographic investigation. A monoclinic crystal form in the space group P2(1) was found to have cell dimensions of a = 122.9 A, b = 101.0 A, c = 95.2 A and beta = 108.3 degrees. The asymmetric unit of the cell contains two dimers of 129 kDa each. A second crystal form, in the triclinic space group P1, has cell dimensions of a = 58.6 A, b = 88.7 A, c = 58.6 A, alpha = 93.3 degrees, beta = 113.8 degrees and gamma = 96.0 degrees, and has one dimer per asymmetric unit.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.     

Crystallographic study of endogenous alpha-amylase inhibitor from wheat

Endogenous alpha-amylase inhibitor from wheat has been crystallized by a microdialysis method. There are two forms of monoclinic crystal in a microdialysis cell with a space group of P2(1). The unit cell dimensions are a = 43.5 A, b = 64.8 A, c = 32.2 A, beta = 113 degrees for the rod-like crystal, and a = 42.5 A, b = 65.2 A, c = 32.2 A, beta = 112 degrees for the plate-like crystal. The former is suitable for structure analysis because it gives the sharp diffraction beyond 2.0 A resolution, and the latter tends to form a twin crystal. A heavy-atom derivative has been successfully prepared with the heavy-atom reagent K2PtCl4, and structure analysis is in progress.     

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA^Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNA^Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA^Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA^Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.     

Crystallization and preliminary X-ray crystallographic analysis of pyridoxine 5'-phosphate oxidase complexed with flavin mononucleotide.

Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.     

Crystal structure of an Escherichia coli tRNA(Gly) microhelix at 2.0 A resolution

tRNA identity elements determine the correct aminoacylation by the cognate aminoacyl-tRNA synthetase. In class II aminoacyl tRNA synthetase systems, tRNA specificity is assured by rather few and simple recognition elements, mostly located in the acceptor stem of the tRNA. Here we present the crystal structure of an Escherichia coli tRNA(Gly) aminoacyl stem microhelix at 2.0 A resolution. The tRNA(Gly) microhelix crystallizes in the space group P3(2)21 with the cell constants a=b=35.35 A, c=130.82 A, gamma=120 degrees . The helical parameters, solvent molecules and a potential magnesium binding site are discussed.     

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.     

A high resolution diffracting crystal form of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase

Three new crystal forms of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase have been produced. The best crystals, obtained after modifying both purification and crystallization conditions, belong to space group P2(1)2(1)2(1) and diffract to 2.7 A. Unit cell parameters are a = 210.4 A, b = 145.3 A and c = 86.0 A (1 A = 0.1 nm), with one dimeric enzyme and two tRNA molecules in the asymmetric unit.     

A trigonal form of the idarubicin:d(CGATCG) complex; crystal and molecular structure at 2.0 A resolution.

The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.     

Crystallization of a ternary complex of lactate dehydrogenase from Bacillus stearothermophilus

Bacillus stearothermophilus lactate dehydrogenase was purified from an overexpressing Escherichia coli cell line. The enzyme has been crystallized in several different forms. All of these crystal forms were grown in the presence of NADH, sodium oxamate and fructose 1,6-bisphosphate. Three crystal forms have been characterized, an orthorhombic P2(1)2(1)2 (type III, a = 86 A, b = 105 A, c = 136 A) and two monoclinic P21 forms (type IV, a = 85 A, b = 118 A, c = 136 A, beta = 96 degrees; type V, a = 112 A, b = 85 A, c = 136 A, beta = 91 degrees). Precession photographs from these crystal forms are very alike, suggesting the molecular packing to be similar in all three forms. The P21 type IV crystals diffract to beyond 2 A spacing and are stable to irradiation with X-rays. A complete medium-resolution (4.7 A) dataset has been collected from a single crystal using synchrotron radiation. Rotation function studies with these data show the two tetramers of the asymmetric unit to be in very similar orientations. Higher-resolution data are being collected.     

Crystallization and preliminary X-ray crystallographic studies of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar)

Crystals of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar) have been grown from ammonium sulphate solution at pH 5.0. Two crystal forms suitable for X-ray structure analysis, obtained from a hanging-drop experiment, have been characterized. Both belong to space-group P22(1)2(1) with cell dimensions a = 39.2 A, b = 62.4 A, c = 84.6 A and a = 31.4 A, b = 74.8 A, c = 83.5 A, for forms I and II, respectively. Intensity data to 1.82 A have been collected for crystal form I on a CAD4 diffractometer, and initial phases have been obtained by molecular replacement methods. The conventional R-factor after two rounds of model building and subsequent refinement is 0.25 for data between 6.0 and 2.0 A. So far no water molecules have been included in the model.     

The structures of crystalline complexes of human serum amyloid P component with its carbohydrate ligand,the cyclic pyruvate acetal of galactose

Two monoclinic (P2(1)) crystal forms of human serum amyloid P component (SAP) in complex with the 4,6-pyruvate acetal of beta-D-galactose (MObetaDG) were prepared. Structure analysis by molecular replacement and refinement at 2.2A resolution revealed that crystal form 1 (a=95.76A, b=70.53A, c=103.41A, beta=96.80 degrees) contained a pentamer in the asymmetric unit with a structure very similar to that of the published search model. The mode of ligand co-ordination was also similar except that four of the five subunits showed bound ligand with an additional H-bond between O1 of the galactose and the side-chain of Lys79. One sub-unit showed no bound ligand and a vacant calcium site close to a crystal contact. The 2.6A resolution structure of crystal form 2 (a=118.60A, b=109.10A, c=120.80A and beta=95.16 degrees ) showed ten sub-units in the asymmetric unit, all with two bound calcium ions and ligand. The most extensive protein-protein interactions between pentamers describe an AB face-to-face interaction involving 15 ion pairs that sandwiches five molecules of bound MObetaDG at the interface.     

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

Crystallization, X-ray diffraction analysis and phasing of carboxylesterase PA3859 from Pseudomonas aeruginosa

We have recently purified an intracellular carboxylesterase encoded by the open reading frame PA3859 of Pseudomonas aeruginosa. Among proteins showing a significant sequence homology with PA3859 the in vivo function is only known for the human acyl-protein thioesterase I that is involved in the deacylation of Galpha proteins. The crystal structure determination of P. aeruginosa carboxylesterase is expected to provide insights into its physiological role. Therefore, the PA3859 gene was cloned and heterologously expressed in Escherichia coli as N-terminally 6xHis tagged recombinant protein. Here, we present the crystallization, X-ray diffraction analysis and phasing of this enzyme. Two crystal forms were obtained by the hanging drop vapor diffusion method. Crystals of form I belong to the space group P2(1) with cell dimensions of a=65.65, b=50.55, c=142.55 A, beta=92.9 degrees and diffracted, upon flash annealing, up to a resolution of 2.9 A. Two dimers are present in the asymmetric unit. Crystals of form II belong to space group P2(1)2(1)2, with unit cell dimensions of a=96.42, b=96.36, c=68.04 A and diffracted up to 2.1 A resolution. One dimer is present in the asymmetric unit.