Crystallization and preliminary X-ray studies of human recombinant interleukin-2

Two different forms of crystals (potentially) suitable for x-ray structure analysis were obtained for recombinant human interleukin-2 (IL-2) using ammonium sulfate as a precipitant in the pH range of 6.3-7.3 (in the case of hexagonal bipyramidal crystals) and 4.5-5.5 (in the case of plate crystals). The hexagonal bipyramidal crystal belongs to a hexagonal space group P6(2)22 or P6(4)22 with a = b = 105.8 A and c = 122.2 A. The crystal diffracts up to 3.4 A resolution and contains 2 or 3 IL-2 molecules in an asymmetric unit. The plate crystal belongs to an orthorhombic space group P2(1)2(1)2 with a = 47.9 A, b = 79.6 A, and c = 31.9 A. The crystal diffracts up to 2.5 A resolution and contains only 1 IL-2 molecule in an asymmetric unit. These facts reconfirmed crystallographically the high homogeneity of the present preparation of human recombinant IL-2.     

Crystallization and structure determination of goat lactoferrin at 4.0 A resolution: a new form of packing in lactoferrins with a high solvent content in crystals

Lactoferrin was purified from fresh samples of goat colostrums, saturated with Fe3+ and CO3(2-) ions and crystallized by microdialysis method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with a=104.6 A, b=153.8 A, c=155.1 A and Z=4. The quality of crystals was poor, thus the intensity data were restricted to 4.0 A resolution only. The structure was determined by molecular replacement method using diferric buffalo lactoferrin as a model. The solution clearly indicated the presence of one molecule in the asymmetric unit, which corresponds to a Vm value of 7.1 A3/Da. The structure was refined with stringent constraints to an R-factor of 0.246 using all the reflections 15,870 to 4.0 A resolution. The overall structure of goat lactoferrin is essentially similar to those of buffalo and bovine lactoferrins. However, the iron-binding environment in goat lactoferrin is somewhat different, in which 2 CO3(2-). ions have low occupancies. The solvent content of approximately 84% was very high in the present case which explains the fragility of the crystals of goat lactoferrin. In a way, it is very surprising that the crystals grow at all, although crystals with solvent as high as 89% have been reported.     

Crystallization and preliminary X-ray investigation of lipoxygenase 1 from soybeans

Soybean lipoxygenase 1 has been crystallized by the vapor diffusion method in 8-10% polyethylene glycol (average Mr 3400), 0.2 M sodium acetate buffer, pH 5.2-5.6, at a protein concentration of 6-12 mg/ml. Microseeding was employed to obtain growth of large single crystals. The crystals, which diffract to at least 2.2-A spacings, are monoclinic and of space group P2(1). Cell constants are: a = 95.4, b = 94.2, and c = 50.4 A and beta = 91.4 degrees. The calculated value of Vm (2.41 A3/Da) is consistent with the probable presence of one molecule of lipoxygenase/crystallographic asymmetric unit (Z = 2).     

Crystallization of a complex between ribonuclease T1 and 2'-guanylic acid

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53% (v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2'-guanylic acid (2'GMP) and 0.02% NaN3 (pH 6.2-7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01-0.05 M sodium acetate, 0.25-0.5% 2'GMP, 0.02% NaN3 and 2-5 mM calcium acetate (pH 4.0-4.4) in the presence of 50-75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and alpha = 4.44 nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2'-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2'GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 x 2'GMP complex is under way.     

Preliminary X-ray diffraction study of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.     

X-ray crystallographic and chromatographic characterization of the crystals of Ca2+-calmodulin complexed with bee venom melittin

Crystals of calmodulin complexed with both Ca2+ and melittin, a peptide from bee venom, have been grown from 2-methyl-2,4-pentanediol solution by using the hanging drop method of vapour diffusion. The crystals belong to space group P2(1)2(1)2(1) with a = 97.3(9) A, b = 56.5(0) A, c = 33.4(9) A and Z = 4. Analyses of the dissolved crystals by high performance liquid chromatography show that the crystals contain a 1:1 complex of calmodulin and melittin. An asymmetric unit contains one such complex and the solvent content of the crystals is 47.5% (v/v).     

Incomplete factorial search for conditions leading to high quality crystals of Escherichia coli cytidine deaminase complexed to a transition state analog inhibitor

We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x-ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.     

Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.     

Crystallization and preliminary X-ray studies of two isolectins from the seeds of Lathyrus ochrus

Two isolectins from the seeds of Lathyrus ochrus, LOL I and LOL II, which specifically bind N-acetyllactosamine, have been crystallized using the hanging-drop method and the interface diffusion method, respectively. In the case of LOL I, 2-methylpentane-2,4-diol, polyethylene-glycol 400 or ammonium sulphate have been used as precipitating agents. The best crystals of LOL I were grown at room temperature from a solution of 40% (v/v) methylpentane diol, 50 mM-Hepes at pH 7.5. LOL II crystals have been grown at room temperature from a solution of 32% (v/v) methylpentane diol, 50 mM-2-(N-morpholino)-ethanesulphonic acid at pH 5.5. X-ray examination of the LOL I and LOL II crystals shows that both are monoclinic, space group P2(1). Their cell dimensions are: LOL I, a = 56.4 A, b = 138.8 A, c = 62.9 A, beta = 91 degrees; and LOL II, a = 54.8 A, b = 71.4 A, c = 105.5 A, beta = 105 degrees. Density measurements of the crystals of LOL I indicate that there are two molecules per asymetric unit (Vm = 2.07 A3/dalton). LOL I crystals diffract strongly up to at least 1.8 resolution. Putative crystals of complexes of LOL I with various glycosides were obtained through co-crystallization under the conditions used for the native protein.     

Single crystals of hydrogenase from Desulfovibrio vulgaris Miyazaki F

The hydrogenase solubilized from the particulate fraction from Desulfovibrio vulgaris Miyazaki F (IAM 12604) has been crystallized. Although the solubilized hydrogenase purified by the previous method (Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S., and Inokuchi, H. (1976) J. Biochem. (Tokyo) 79,661-671) revealed a single band upon disc electrophoresis, it could not be crystallized. The apparently homogeneous hydrogenase has been separated into three components of similar molecular weights by high performance liquid chromatography on DEAE-Toyopearl. Each hydrogenase component was successfully crystallized by means of the vapor diffusion method with polyethylene glycol or 2-methyl-2,4-pentanediol as a precipitating agent. Seeding procedure is necessary to grow an x-ray grade crystal. Preliminary x-ray experiments reveal that crystals grown from one component are in space group of P2(1)2(1)2(1) with a = 102.1(1), b = 126.8 (3), and c = 66.9(1) A. The unit cell volume of 8.66 X 10(5) A3 suggests that it contains one molecule/asymmetric unit (Vm = 2.43). The crystals grown from another component are in the same space group with a = 99.6(1), b = 126.8(3), c = 66.9(1) A, and the unit cell volume is 8.45 X 10(5) A3 (Vm = 2.37). The crystals diffract more than 2.5 A and are suitable for complete crystal analysis. Up to 4 A resolution native data have been collected on a diffractometer.     

Crystallization and preliminary x-ray investigation of the regulatory subunit of cAMP-dependent protein kinase

Crystals of type I cAMP-dependent protein kinase regulatory subunit have been grown from solutions of ammonium sulfate. The crystals are square bipyramids, space group P4(1)2(1)2 (P4(3)2(1)2), with a = b = 106.9 +/- 0.6 A and c = 212.4 +/- 1.0 A. There are two dimers of the regulatory subunit/crystallographic asymmetric unit. The crystals are stable for 3-4 days in the x-ray beam and diffract to at least 3.5-A resolution.     

Antiparallel beta-sheets in the crystal structure of the heptapeptide Met-Glu-His-Phe-Arg-Trp-Gly (ACTH 4-10)

The conformation of the molecules in ACTH 4-10 has been determined as part of a study of the conformations of the biologically active N-terminal fragments of the adrenocorticotropic hormone (ACTH). ACTH 4-10 crystallizes in two different superstructures. The substructure considered in the present work, is monoclinic, space group C2, a = 25.75(1) A, b = 27.78(1) A, c = 20.35(1) A, beta = 114.0(1) degrees, Z = 8 molecules ACTH 4-10 plus 22 weight per cent solvent. The crystals contain antiparallel beta-sheets, the orientations of the side groups are not found, because of disorder. The present crystal structure and those of other linear oligopeptides emphasize that antiparallel beta-sheets are energetically favourable. It is very unlikely, however, that the ACTH 4-10 crystals contain the molecules in their biologically active form.     

Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli

Ribonuclease H (RNase H) from Escherichia coli is an endonuclease that specifically degrades the RNAs of RNA:DNA hybrids. The enzyme is a single polypeptide chain of 155 amino acid residues, of which 4 are methionines. To solve the crystallographic three-dimensional structure of E. coli RNase H by the multi-wavelength anomalous diffraction technique, we have constructed methionine auxotrophic strains of E. coli that overexpress selenomethionyl RNase H. MIC88 yields about 10 mg of selenomethionyl RNase H per liter of culture, which is comparable to the overexpression of the natural recombinant protein. We have purified both proteins to homogeneity and crystallized them isomorphously in the presence of sulfate. These are Type I crystals of space group P2(1)2(1)2(1) with the cell parameters a = 41.8 A, b = 86.4 A, c = 36.4 A, one monomer per asymmetric unit, and approximately 36% (v/v) solvent. Crystals of both proteins diffract to beyond 2-A Bragg spacings and are relatively durable in an x-ray beam. On replacement of sulfate with NaCl, crystals of natural RNase H grow as Type I' (very similar to Type I) at pH between 7.0 and 8.0; at pH 8.8, crystals of Type II are obtained in space group P2(1)2(1)2(1) with a = 44.3 A, b = 87.3 A, and c = 35.7 A. Type II crystals can be converted to Type I by soaking in phosphate buffer. RNase H crystals of Type II have also been reported by Kanaya et al. (Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264, 11546-11549).     

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

Crystallization and preliminary crystallographic data of chicken gizzard G-actin . DNase I complex and Physarum G-actin . DNase I complex.

Smooth muscle G-actin from chicken gizzard and Physarum plasmodium G-actin both interact with DNase I and form 1 : 1 complexes. These complexes were crystallized by using polyethylene glycol 6000 as a precipitant. Both crystals belong to the same orthorhombic space group P2(1)2(1)2(1). The cell dimensions of chicken gizzard G-actin.DNase I complex are a=42.00 +/- 0.07 A, b=225.3 +/- 0.4 A, and c=77.4 +/- 0.1 A, while those of Physarum G-actin.DNase I complex are a=42 A, b=221 A, and c=77 A.     

Preliminary X-ray studies of the tetra-heme cytochrome c3 and the octa-heme cytochrome c3 from Desulfovibrio gigas

A tetra-heme and an octa-heme cytochrome c3 from the sulfate bacterium Desulfovibrio gigas have been crystallized. Diffraction quality crystals of the tetra-heme cytochrome are obtained from solution by the addition of polyethylene glycol at pH 6.5. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell parameters a = 42.27 A, b = 52.54 A and c = 52.83 A. The octa-heme cytochrome crystals develop from low ionic strength solutions of phosphate or Tris-Cl in the pH range 6.2-7.6. The crystals belong to the trigonal system, space group P3(1) or the enantiomorph P3(2), with unit cell parameters a = b = 57.4 A, c = 97.3 A, gamma = 120 degrees. Single crystal diffraction studies of the structures of these two low-potential cytochromes are in progress.     

Crystallization of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.     

Crystallization and preliminary X-ray diffraction studies of a complex between interleukin-2 and a soluble form of the p55 component of the high affinity interleukin-2 receptor

Human recombinant interleukin-2 (IL-2) and a soluble recombinant form of the human p55 (Tac antigen) component of the IL-2 receptor (IL-2R) have been cocrystallized in 1.7-1.8 M ammonium sulfate, in the pH range 7.0-8.2. Variously glycosylated forms of both receptor and ligand can be cocrystallized under those conditions. The best crystals of the putative receptor-ligand complex involve the enzymatically desialylated receptor and unglycosylated IL-2. These crystals belong to the trigonal space group P3(1)2(1) or its enantiomorph, with unit cell dimensions a = b = 91 A and c = 119 A, and diffract to 3.5 A resolution. There is one receptor-ligand complex asymmetric unit, with a Matthews coefficient of 2.7, assuming the presence of one IL-2 molecule-receptor molecule. Interestingly, in addition to IL-2 (Mr = 14,000), the p55 IL-2 receptor (Mr = 44,000) and two fragments of the receptor, of apparent Mr = 35,000 and 25,000, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crystals are enriched in a reducible dimeric form of the desialylated receptor (apparent Mr = 90,000), as compared with protein solution from which the crystals grow. The overall amino acid content in the crystals is consistent with a 1:1 ratio of receptor to ligand. A native data set has been collected on a multiwire area detector and the search for suitable heavy atom derivatives is in progress.     

Crystallization of and preliminary crystallographic data for allosteric L-lactate dehydrogenase from Bifidobacterium longum

L-Lactate dehydrogenase from Bifidobacterium longum aM101-2 was overexpressed in Escherichia coli and then purified. The enzyme was crystallized from a polyethylene glycol 6000 solution by the hanging drop vapor diffusion method. Crystals grown in the presence of NADH (type II), both NADH and oxamate (type III), and NADH, oxamate, and FBP (type IV) were analyzed. All three crystal forms belong to the orthorhombic system, space group P2(1)2(1)2. The cell dimensions of the type II crystals were a = 106.2 A, b = 131.6 A, and c = 63.8 A. Those of the type III and type IV crystals were a = 106.4 A, b = 131.4 A, and c = 63.8 A. The type III crystals diffract X-rays to beyond 2.5 A spacing. The type II and type III crystals were stable as to X-ray irradiation.     

Antiparallel pleated beta-sheets observed in crystal structures of N,N-bis(trichloroacetyl) and N,N-bis(m-bromobenzoyl) gramicidin S.

Despite intensive efforts, the structures of gramicidin S (GS) [cyclo(-Val-Orn-Leu-d-Phe-Pro-)(2)] and its analogues have not been elucidated by the X-ray diffraction method, except for the GS-urea complex (Hull et al., Nature 275, 206-207, 1978; Tishchenko et al., Acta Cryst. D53, 151-159, 1997). We focused on the acetylation of GS to obtain suitable crystals for X-ray diffraction. The amino groups of Orn residues were capped with trichloroacetic and m-bromobenzoic acids. Both trichloroacetyl and m-bromobenzoyl GSs (TcGS and BzGS, respectively) are hydrophobic and their properties are similar to those of acetyl-GS (AcGS). Although it is well known that AcGS yields hexagonal crystals, TcGS and BzGS yield monoclinic and orthorhombic crystals in aqueous dimethylformamide solution, respectively. Their cell volumes were approximately one-fourth or one-eighth of the hexagonal cell volume. The crystal structures of TcGS and BzGS were determined as the first examples of acetylated GS analogues: TcGS, C(64)H(90)N(12)O(12)Cl(6). 3(C(3)H(7)NO), M(r) = 1651.47, monoclinic, P2(1), a = 15.4366(6) A, b = 18.5312(4) A, c = 16.4774(6) A, beta = 14.160(2) degrees, V = 4300.6(2) A(3), Z = 2; and BzGS, C(64)H(98)N(12)O(12)Br(2). 1.54(H(2)O), M(r) = 1535.21, orthorhombic, P2(1)2(1)2(1), a = 16.748(10) A, b = 18.834(5) A, c = 28.558(10) A, V = 9008(7) A(3), Z = 4. Both these peptide molecules formed an antiparallel pleated beta-sheet, and pseudo twofold symmetries existed in the repeated sequence. beta-Turns formed at the fragments of d-Phe-Pro were classified into type II' based on their characteristics. The peptide conformations of TcGS and BzGS were similar to each other, and these structural features agreed with those of structures proposed by the previous studies.