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 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 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.     

Crystallization and subunit structure of canine myeloperoxidase

Three crystal forms of canine myeloperoxidase are described. An orthorhombic form in space group P2(1)2(1)2(1) has unit cell dimensions: a = 108.3 A (1 A = 0.1 nm) b = 205.9 A and c = 139.9 A. A trigonal form in space group P3(1)21 or P3(2)21 has unit cell dimensions: a = b = 138.9 A and c = 145.2 A. A monoclinic form in space group C2 has unit cell dimensions: a = 117.2 A, b = 96.9 A, c = 131.4 A and beta = 116.3 degrees. Unusual features in the diffraction patterns of the monoclinic form place restrictions on the molecular packing in the crystal. The proposed model for the molecular packing requires that the myeloperoxidase molecule consist of two identical or near-identical halves. In the intact molecule these halves may be related either by a crystallographic dyad axis or by an approximate dyad axis in which one subunit is translated relative to the other by 3.2 A along the symmetry axis. The trigonal crystal form appears most suitable for high-resolution X-ray structural analysis.     

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.     

Crystallization and preliminary X-ray diffraction analysis of <Emphasis Type="Italic">E. coli</Emphasis> arginyl-tRNA synthetase in complex form with a tRNA<Superscript>Arg</Superscript>

Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA(Arg) (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 A and 94% completeness has been obtained, with unit cell parameters a = b = 98.0 A, c = 463.2 A, and alpha = beta = 90 degrees , gamma = 120 degrees , being different from a = b = 110.8 A, c = 377.8 A for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.     

Crystallization of the met repressor from Escherichia coli

The met repressor from Escherichia coli has been crystallized in space group P21, with unit cell dimensions a = 35.6 A, b = 62.6 A, c = 44.5 A, beta = 102.4 degrees and one aporepressor dimer per asymmetric unit. Preliminary X-ray diffraction photographs show measurable intensities to beyond 1.5 A resolution, and the crystal form is ideally suited to high-resolution crystallographic analysis (1 A = 0.1 nm).     

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 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 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 and preliminary crystallographic study of DNA polymerase from Pyrococcus furiosus

A new member of archaeal DNA polymerase from Pyrococcus furiosus was crystallized. Diffraction data to 3.1 A of the selenomethionine-derivatized crystal were collected, and preliminary crystallographic study has been completed. The crystal belongs to the space group C2 with unit cell parameters of a = 93.2 A, b = 124.9 A, c = 87.7 A, alpha = 90 degrees , beta = 109.7 degrees , and gamma = 90 degrees . Assuming the presence of one molecule in the asymmetric unit, the solvent content of the crystal is estimated to be 54%, corresponding to a Matthews coefficient V(M) of 2.7A (3) Da(-1).     

X-ray grade crystals of the enzymatic fragment of diphtheria toxin

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.     

Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein.

The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.     

Crystal structure of a light-harvesting protein C-phycocyanin from Spirulina platensis

The crystal structure of C-phycocyanin, a light-harvesting phycobiliprotein from cyanobacteria (blue-green algae) Spirulina platensis has been solved by molecular replacement technique. The crystals belong to space group P2(1) with cell parameters a = 107.20, b = 115.40, c = 183.04 A; beta = 90.2 degrees. The structure has been refined to a crystallographic R factor of 19.2% (R(free) = 23.9%) using the X-ray diffraction data extending up to 2.2 A resolution. The asymmetric unit of the crystal cell consists of two (alphabeta)6-hexamers, each hexamer being the functional unit in the native antenna rod of cyanobacteria. The molecular structure resembles that of other reported C-phycocyanins. However, the unique form of aggregation of two (alphabeta)6-hexamers in the crystal asymmetric unit, suggests additional pathways of energy transfer in lateral direction between the adjacent hexamers involving beta155 phycocyanobilin chromophores.     

Crystallization of an odorant-binding protein from cow nasal mucosa

The first odorant-binding protein isolated from mammalian nasal mucosa is a dimer of subunits of identical molecular weight (19,000) that specifically binds bell pepper odorants, "green" smelling compounds. The protein can be purified in milligram quantities from tissue extractions by sequential use of a silica based anion exchange column and Mono-P chromatofocussing column. In the presence of the binding compound 2-isobutyl-3-methoxypyrazine and of the organic solvent 2-methyl-2,4-pentanediol (17%, v/v), the protein crystallizes in the monoclinic space group P2(1), with unit cell constants a = 54.3 A, b = 66.7 A, c = 41.5 A, beta = 97.2 degrees. From consideration of the crystal packing densities compatible with its unit cell, it can be concluded that two subunits of 19,000 Mr each are present in the asymmetric unit. The diffraction pattern on still photographs of this crystal form of the protein extends to 2.5 A resolution and allows for a detailed crystallographic investigation.     

Crystallization and preliminary X-ray studies of the pectate lyase from Bacillus subtilis.

The pectate lyase (EC 4.2.2.9) from Bacillus subtilis has been crystallized. Crystals of form 1, grown by the hanging drop method using polyethylene glycol as precipitant, diffract to at least 2.4 A resolution. They belong to the spacegroup P2(1) with a = 132.9 A, b = 41.2 A, c = 156.8 A and beta = 114.9 degrees with probably four molecules in the asymmetric unit. A second crystal form grown from 2-methyl-2,4-pentandiol also belongs to the spacegroup P2(1) with a = 55.0 A, b = 88.1 A, c = 50.2 A and beta = 109.0 degrees. These crystals diffract to at least 2.0 A and have one molecule in the asymmetric unit. Both crystal forms are suitable for the determination of high-resolution structures.     

Trigonal crystals of porcine mitochondrial aspartate aminotransferase

Crystals suitable for X-ray analysis of porcine mitochondrial aspartate aminotransferase in the closed conformation were obtained after the apoenzyme was reconstituted with N-5'-phosphopyridoxyl-L-aspartate, an inhibitor in which the cofactor is covalently bound to the substrate. This results in a crystal form that has not been encountered previously in studies of aspartate aminotransferases. The crystals belong to the trigonal space group P3121 (or the enantiomeric P3221) with unit cell dimensions alpha = b = 202.0 A, c = 58.0 A, alpha = beta = 90 degrees, gamma = 120 degrees and contain one dimer in the asymmetric unit.     

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.     

D-alanine-D-alanine ligase (ADP) from Salmonella typhimurium. Overproduction, purification, crystallization and preliminary X-ray analysis

The ddlA gene from Salmonella typhimurium coding for D-alanine-D-alanine ligase (ADP-forming) has been subcloned behind the tac promotor in the plasmid pKK223-3, with expression in Escherichia coli JM105. The overexpression system yields 58 mg of active enzyme from 12 g of wet cell paste after 40-fold purification to homogeneity. 5,5'-Dithiobis-(2-nitrobenzoic acid) titrations indicate that all four cysteine residues exist as free thiols. Two crystal forms of the 39,300 Mr enzyme have been produced. A tetragonal form grows at 21 degrees C from 10 to 15% (w/v) polyethylene glycol 8000 in space group P4(1)2(1)2, with two molecules in the asymmetric unit; it has cell constants a = b = 83.8 A, c = 220.0 A, and diffracts to 2.9 A. A monoclinic form grows from 30% (w/v) ammonium sulfate in space group P2(1), with two molecules in the asymmetric unit; it has cell constants a = 60.4 A, b = 102.1 A, c = 64.3 A, beta = 115.7 degrees, and diffracts to 2.2 A resolution.     

Crystallization of a photosensitive nitrile hydratase from Rhodococcus sp. N-771.

A photosensitive nitrile hydratase from Rhodococcus sp. N-771 has been crystallized in two different crystal forms in its inactive form. One crystal form belongs to an orthorhombic space group P2(1)2(1)2 with unit cell dimensions of a = 117.4 A, b = 145.7 A and c = 52.1 A, and the other form belongs to a hexagonal space group P6(3)22 with unit cell dimensions of a = 110.2 A and c = 412.1 A.