Preliminary x-ray investigation of an orthorhombic crystal form of human plasma albumin.

An orthorhombic form of single crystals of human plasma albumin, suitable for x-ray diffraction studies, has been grown with ammonium sulfate from protein solutions purified from fresh frozen single donor plasma as well as from a commercial sample of plasma albumin. The space group is P2(1)2(1)2 with 12 molecules in the unit cell. The cell dimensions are: a = 133.3 +/- 1.2 A, b = 274.8 +/- 3.3 A,, and c = 58.02 +/- 0.02 A.     

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.     

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.     

Preliminary X-ray diffraction analysis of HhaII endonuclease-DNA cocrystals

HhaII restriction endonuclease purified from an overproducing recombinant E. coli clone has been cocrystallized with a heptanucleotide duplex, d-GGAGTCC:GGACTCC. The cocrystals are monoclonic and belong to the space group C2. The unit cell dimensions are a = 199.0 +/- 1.0 A, b = 100.0 +/- 0.5 A, c = 80.3 +/- 0.4 A, and beta = 101.0 +/- 1.0 degrees. There appear to be two dimers per asymmetric unit and the crystals diffract to 4-A resolution.     

X-ray diffraction analysis on single crystals of recombinant Escherichia coli ornithine transcarbamoylase

Single crystals of recombinant Escherichia coli ornithine transcarbamoylase suitable for x-ray analysis have been grown from polyethylene glycol and 2-methyl-2,4-pentanediol. The space group has been determined as P3(1) or P3(2), with one protein trimer of three identical 36.8-kDa subunits in the asymmetric unit. The unit cell dimensions are a = b = 105.1 A and c = 87.8 A. The crystals diffract well to 3-A resolution and are quite resistant to radiation damage. Single crystals have also been grown of a genetically engineered site-specific mutant for which the replacement of an arginine (Arg-57) to a glycine has been shown to not only drastically affect the enzyme activity but also its kinetic mechanism (Kuo, L. C., Miller, A. W., Lee, S., and Kozuma, C. (1988) Biochemistry 27, 8823-8832). The crystals of the Arg-57----Gly mutant protein are isomorphous to those of the wild type. Crystal soaking experiments using both wild-type and Arg-57----Gly crystals in the presence of various ligands have provided evidence of specific conformational changes upon substrate binding which supports our previous kinetic and spectroscopic observations.     

Crystal parameters and molecular replacement of an anticholera toxin peptide complex.

TE33 is an Fab fragment of a monoclonal antibody raised against a 15-residue long peptide (CTP3), corresponding in sequence to residues 50-64 of the cholera toxin B subunit. Crystals of the complex between TE33 and CTP3 have been grown from 20% (w/v) polyethylene glycol-8000 at pH 4.0. The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 104.15, b = 110.61, and c = 40.68 A. X-Ray data have been collected to a resolution of 2.3 A. The asymmetric unit contains one molecule of Fab and one molecule of CTP3. The presence of CTP3 has been demonstrated by fluorescence quenching of the dissolved crystal after X-ray data collection. A molecular replacement solution was found based on the coordinates of DB3, an antiprogesterone Fab fragment.     

Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase

Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes.     

Two-dimensional crystal structures of protein kinase C-delta, its regulatory domain, and the enzyme complexed with myelin basic protein

Two-dimensional crystals of protein kinase C (PKC) delta, its regulatory domain (RDdelta), and the enzyme complexed with the substrate myelin basic protein have been grown on lipid monolayers composed of phosphatidylcholine: phosphatidylserine: diolein (45:50:5, molar ratio). Images have been reconstructed to 10-A resolution. The unit cells of all three proteins have cell edges a = b and interedge angle gamma = 60 degrees. RDdelta has an edge length of 33 +/- 1 A, and its reconstruction is donut shaped. The three-dimensional reconstructions from the PKCdelta C1b crystal structure () can be accommodated in this two-dimensional projection. Intact PKCdelta has an edge length of 46 +/- 1 A in the presence or absence of a nonhydrolyzable ATP analog, AMP-PnP. Its reconstruction has a similar donut shape, which can accommodate the C1b domain, but the spacing between donuts is greater than that in RDdelta; some additional structure is visible between the donuts. The complex of PKCdelta and myelin basic protein, with or without AMP-PnP, has an edge length of 43 +/- 1 A and a distinct structure. These results indicate that the C1 domains of RDdelta are tightly packed in the plane of the membrane in the two-dimensional crystals, that there is a single molecule of PKCdelta in the unit cell, and that its interaction with myelin basic protein induces a shift in conformation and/or packing of the enzyme.     

Single crystals of bacteriophage T7 RNA polymerase

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.     

Molecular symmetry of fructose-1,6-diphosphatase by X-ray diffraction analysis.

Large single crystals of rabbit liver fructose-1,6-diphosphatase suitable for a high resolution structure analysis have been grown from polyethylene glycol. The space group of these crystals is I222 with a = 75 A, b = 81 A, and c = 132 A and there are 2 tetrameric molecules in the unit cell. These crystals have one protein subunit as the crystallographic asymmetric unit and establish point group symmetry 222 as the molecular symmetry.     

Single crystals of chitosan

Lamellar single crystals of chitosan were prepared at 125 degrees C by adding ammonia to a low DP fraction of chitosan dissolved in water. The crystals gave sharp electron diffraction diagrams which could be indexed in an orthorhombic P2(1)2(1)2(1) unit cell with a = 8.07 A, b = 8.44 A, c = 10.34 A. The unit cell contained two anti-parallel chitosan chains and no water molecules. It was found that cellulose microfibrils from Valonia ventricosa could act as nuclei for inducing the crystallization of chitosan on cellulose. This produced a shish-kebab morphology.     

Characterization of bacterial photosynthetic reaction center crystals from Rhodopseudomonas sphaeroides R-26 by X-ray diffraction

An orthorhombic crystal form (P2(1)2(1)2(1)) of the reaction center from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 has been characterized. The crystals were grown from polyethylene glycol; the unit cell dimensions are a = 142.2 A, b = 139.6 A, and c = 78.7 A; and they contain one reaction center in each crystallographic asymmetric unit. The crystals diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.     

Crystallization of an endochitinase from Hordeum vulgar L. seeds.

Higher plants contain several constitutively expressed proteins for protection against infections by viruses, bacteria and fungi. Here we report the crystallization of a polypeptide with antifungal activity, a 26,000 dalton endochitinase from barley (Hordeum vulgare L.) seeds, in a form suitable for high-resolution X-ray analysis. Crystals were grown by vapor diffusion under several different conditions. The best crystals, obtained with ammonium sulfate as the precipitant, belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2), with cell dimensions a = b = 62.9 A and c = 96.0 A. The cell dimensions are consistent with one endochitinase molecule per asymmetric unit, and the crystals diffract to at least 2.0 A resolution.     

Preparation of crystals of chicken cytosolic aspartate amino- transferase, suitable for high resolution x-ray structural analysis

The crystals of cytosolic chicken aspartate aminotransferase were grown from polyethylene glycol solutions. Two of the four crystal modifications obtained diffract to 1.8 A resolution. The crystals of the free holoenzyme belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 56.9, b = 126.9, c = 124.6 A. The crystals of the enzyme-maleate complex belong to the same space group with slightly different unit cell dimensions of a = 56.5, b = 126.1, c = 124.6 A. The influence of ions of several divalent metals, dioxane and non-ionic detergent beta-octylglucoside on crystallization have been investigated. The best crystals were obtained in the presence of Mg2+ ions. These crystals were used for data collection on the diffractometer.     

Isolation, crystallization and preliminary X-ray diffraction data of human progastricsin

Human progastricsin, a zymogen of one of the gastric aspartic proteinases, was isolated and crystallized. The crystals belong to the tetragonal space group P4(2)2(1)2, and have unit cell dimensions a = b = 105.5 +/- 0.1 A, c = 70.6 A. The native crystals of progastricsin diffract X-rays at least to 2.5 A and are suitable for a high-resolution X-ray analysis.     

Crystallization and preliminary X-ray crystallographic properties of Hsc20, a J-motif co-chaperone protein from Escherichia coli.

Hsc20 is a 20-kDa auxiliary protein that functions with the molecular chaperone Hsc66 in Escherichia coli. Crystals of Hsc20 suitable for X-ray diffraction analysis were grown using the hanging drop vapor diffusion technique in polyethylene glycol 400 containing dioxane as an additive to slow growth. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions a = 125.4 A, b = 71.9 A, c = 68.8 A, and beta = 97.0 degrees. The crystals diffract to a minimum d-spacing of approximately 2.5 A resolution, and a native data set was collected to 2.7 A. The results of a self-rotation function analysis revealed threefold symmetry, suggesting three molecules of Hsc20 in the asymmetric unit and, hence, 12 molecules in the unit cell; this corresponds to a Vm value of 2.6 A3/Da and a solvent content of approximately 53% in the crystals. Structure determination by isomorphous replacement is in progress.     

Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.     

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.     

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).     

Crystallization of a high potential iron-sulfur protein from the halophilic phototrophic bacterium Ectothiorhodospira halophila

Crystals of the high-potential iron-sulfur protein from Ectothiorhodospira halophila strain BN 9626 have been grown from 3.4 to 3.5 M ammonium sulfate solutions at pH 7.5. The crystals belong to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. There are 2 molecules/asymmetric unit. The crystals diffract to at least 1.8 A, are stable in the x-ray beam, and are suitable for a high resolution x-ray crystallographic analysis.