Expression, purification, crystallization and preliminary crystallographic analysis of the human intracellular chloride channel protein CLIC4

The human chloride intracellular channel protein CLIC4 has been crystallized by the hanging-drop vapour-diffusion technique using trisodium citrate as the precipitant. The best crystals were obtained by the microseeding method. The crystals diffracted to 2.2 A resolution and were found to belong to space group P121, with unit-cell parameters a = 73.19, b =86.05, c = 73.38 A, beta = 112.99 degrees and three molecule per asymmetric unit.     

Crystallization and preliminary X-ray crystallographic studies of Thermus thermophilus HB8 MutM protein involved in repairs of oxidative DNA damage

MutM protein, which removes the oxidatively damaged DNA base product, 8-oxoguanine (GO), has been crystallized by means of a hanging-drop vapor-diffusion procedure using polyethyleneglycol monomethylether 2000 as a precipitant in 2-(cyclohexylamino) ethanesulfonic acid (CHES) buffer, pH 9.8. The diffraction data derived from oscillation photographs indicate that the crystals belong to the monoclinic system and space group P2(1). The crystals have unit-cell dimensions of a = 45.4 A, b = 62.0 A, c = 99.7 A, and beta = 90.8 degrees. Assuming that the asymmetric unit contains two molecules, the Vm value was calculated to be 2.35 A(3).Da(-1). The crystals diffracted X-rays to at least 2.1 A resolution and were suitable for high-resolution X-ray crystal structure determination.     

Crystallization of halorhodopsin from Halobacterium sp. shark

The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.     

Crystallization and preliminary X-ray diffraction analysis of the extracellular domain of the cell surface antigen CD38 complexed with ganglioside

The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X-ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da.     

Crystallization and preliminary X-ray diffraction studies of a flavoprotein, FP390, from a luminescent bacterium, Photobacterium phosphoreum.

A flavoprotein, FP390, obtained from a luminescent bacterium, Photobacterium phosphoreum, in the purification of luciferase has been crystallized by the vapor-diffusion procedure. Crystals obtained from polyethylene glycol 4000 solutions, whose X-ray photographs show powder diffraction patterns, were unsuitable for further crystallographic work. However, tetragonal crystals grown from potassium phosphate solution well diffracted X-rays beyond 3 A resolution. The space group of this crystal is P4(1)22 or P4(3)22 with unit-cell dimensions of a = b = 76.8 and c = 241 A. Assuming two or three molecules in an asymmetric unit, the value for the crystal volume per unit molecular mass, Vm, is calculated as 3.3 or 2.2 A3/Da, respectively. A total of 13,555 independent reflections for the native crystal was collected up to 3 A resolution using a Weissenberg camera attached to the synchrotron radiation source, the merging R factor being 0.077 for 79,335 measurements.     

Crystallization and preliminary diffraction studies of malate dehydrogenase from Streptomyces aureofaciens

Purified malate dehydrogenase (MDH) of Streptomyces aureofaciens was crystallized either in the absence or in the presence of NADH or NADPH coenzymes by hanging-drop vapour-diffusion method. An X-ray study has shown, that MDH crystals belong to space group C222(1) with unit-cell parameters a = 53.2 A, b = 104.6 A, c = 520.0 A, alpha = beta = gamma = 90( degrees ), MDH-NADH crystals to space group C2 with unit-cell parameters a = 51.5 A, b = 51.5 A, c = 256 A, alpha = beta = gamma = 90( degrees ), and MDH-NADPH crystals to space group C222(1) with unit-cell parameters a = 72, A b = 72 A, c = 520 A, alpha = beta = gamma = 90( degrees ). The crystal of native MDH diffracted to 2.1 A resolution.     

Crystallization and preliminary x-ray analysis of complexes of porcine pancreatic elastase with two natural inhibitors

Two different natural protease inhibitors, the squash inhibitor MCEI III and the third domain of turkey ovomucoid inhibitor OMTKY3, were crystallized in complexes with porcine pancreatic elastase (PPE). About 700 conditions were screened altogether. Crystals of the complex between MCEI III and PPE were grown in citrate buffer with and without ammonium acetate. X-ray diffraction data were collected to 1.9 angstroms resolution at room temperature using synchrotron radiation. The crystals belong to space group P2(1), with unit-cell parameters a = 49.17, b = 44.59, c = 67.08 angstroms, beta = 110.97 degrees. Crystals of the OMTKY3/PPE complex were obtained in the presence of ammonium sulfate, MES buffer and polyethylene glycol monomethylether (PEG). These crystals of this complex diffracted to 2.1 angstroms resolution and belongs to space group I222, with unit-cell parameters a = 84.58, b = 84.61, c = 89.92 angstroms and diffracted to 2.2 angstroms resolution. The diffraction data were collected using a conventional rotating anode X-ray generator at room temperature. In both cases the presence of inhibitor in the crystals was confirmed by crystallography.     

Crystallization and preliminary X-ray crystallographic studies of bovine heart mitochondrial cytochrome bc1 complex.

Cytochrome bc1 complex (ubiquinol:ferricytochrome c oxidoreductase, EC. 1.10.2.2) from bovine heart mitochondria was crystallized by a batchwise method from protein solution containing sucrose monolaurate using polyethylene glycol-4000 as a precipitant. The red parallelepiped crystals grew to a size of approximately 1 mm x 1 mm x 1 mm. The crystalline protein showed enzymic activity catalyzing electron transfer from ubiquinol-2 to cytochrome c. The subunit composition and absorption spectrum of the crystalline enzyme were identical to those reported previously for the enzyme in solution. The crystal diffracted X-rays to 7.5 A resolution. The diffraction pattern indicated a monoclinic form, space group P2(1), and unit-cell constants of a = 196 A, b = 179 A, c = 253 A and beta = 97 degrees. Most probably four functional units are present in an asymmetric unit.     

Preliminary X-ray crystallographic study of lysozyme produced by Streptomyces globisporus

Lysozyme from Streptomyces globisporus has been crystallized in a form suitable for X-ray structure analysis using ammonium sulfate as a precipitant. The crystals are hexagonal, space group P6(1)22 (P6(5)22) with unit cell dimensions: a = b = 129 A, c = 143 A. There are three or four molecules per asymmetric unit. The crystals diffract X-rays to at least 3.0 A resolution.     

Crystallization and preliminary X-ray investigation reveals that tumor necrosis factor is a compact trimer furnished with 3-fold symmetry

Crystals of recombinant human tumor necrosis factor produced by Escherichia coli have been obtained under different conditions. Crystals suitable for X-ray studies are produced by a vapor diffusion technique using sodium phosphate as both precipitant and buffer at pH 6.5. The crystals belong to the cubic space group, P2(1)3 with unit cell dimensions a = b = c = 95.7 A (1 A = 0.1 nm). Preliminary photography reveals that the crystals are moderately stable to X-rays and diffract to at least 3 A resolution. The diffraction data for native crystals have been collected on a diffractometer at 3 A resolution. Another crystal form, which appeared in a solution containing sodium phosphate at pH 8.0, has the trigonal space group P3 with unit cell dimensions a = b = 63.8 A and c = 54.4 A, and produces measurable reflections to a resolution of 3 A. Hexagonal crystals also have been obtained by the use of polyethylene glycol as precipitant in the range pH 7.6 to 8.0; however, the crystals are fragile and unstable to X-rays. Conservation of 3-fold symmetry in the different crystal forms obtained could reflect the ability of tumor necrosis factor molecules to form trimers in solution and probably the nature of binding of the molecules to cellular receptors.     

Crystallization and preliminary X-ray diffraction studies of aspartic proteinase from Irpex lacteus.

Crystals of ILAP (Irpex lacteus aspartic proteinase) have been obtained by the hanging drop method using ammonium sulfate as a precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 54.5 A, b = 79.6 A, c = 37.5 A, beta = 96.8 degrees. The crystals are quite stable to X-rays and diffract beyond 1.9 A resolution. There is one molecule in the asymmetric unit.     

Preliminary crystallographic analysis of cardiotoxin V with major fusion activity from Taiwan cobra (Naja naja atra) venom

Crystals of a cardiotoxin from Taiwan cobra venom have been obtained by the vapor diffusion method using methyl pentanediol as precipitant. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 47.5 A, c = 111.3 A, alpha = beta = 90 degrees and gamma = 120 degrees and diffract to a resolution of 2.2 A. There is one molecule per asymmetric unit and the solvent content is estimated to be 53%.     

Preliminary x-ray crystallographic analysis of centrin from ciliate Euplotes octocarinatus

Centrins are four-EF-hand Ca(2+)-binding proteins, which belong to the CaM super family. The centrin from ciliate Euplotes Octocarinatus has been expressed in Escherichia coli, purified and crystallized using the hanging-drop method. Rod-like crystals were grown and diffracted to 2.0 angstroms. The crystals belong to space group P2(1)2(1)2(1) and the unit-cell parameters are a=34.442 angstroms, b=48.954 angstroms, c=72.583 angstroms.     

Cloning,expression, crystallization,and preliminary X-ray analysis of recombinant mouse lipocalin-type prostaglandin D synthase,a somnogen-producing enzyme

Lipocalin-type prostaglandin D synthase is the key enzyme for the production of prostaglandin D(2), a potent endogenous somnogen, in the brain. We cloned, produced, and crystallized the native enzyme and selenomethionyl Cys(65)Ala mutants of the recombinant mouse protein by the hanging drop vapor-diffusion method with both malonate and citrate as precipitants. The native crystals obtained with malonate belong to orthorhombic space group P2(1)2(1)2(1) with lattice constants a = 46.2, b = 66.8, and c = 105.3 A. The selenomethionyl crystals obtained with citrate belong to orthorhombic space group C222(1) with lattice constants a = 45.5, b = 66.8, and c = 104.5 A. The native crystals diffracted beyond 2.1 A resolution.     

Crystallographic studies on a family B DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1.

A hyperthermostable family B DNA polymerase from the hyperthermophilic archaeon, Pyrococcus kodakaraensis strain KOD1, has been crystallized by the hanging-drop vapor diffusion method at 293 K with 2-methyl-2,4-pentanediol as the precipitant. The diffraction pattern of a crystal extends to 3.0 A resolution, and two full sets of 3.0 A resolution diffraction data for native crystals were successfully collected at 290 K and 100 K upon exposure to synchrotron radiation at KEK-PF, Japan. The crystals belong to the space group, P212121, with unit-cell dimensions of a = 112.8, b = 115.4, and c = 75.4 A at 290 K, and a = 111.9, b = 112.4, and c = 73.9 at 100 K. Structural analysis by means of the multiple isomorphous replacement method is now in progress.     

New crystal form of recombinant murine interferon-beta

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.     

Crystallization and preliminary X-ray analysis of a DNA primase from hyperthermophilic archaeon Pyrococcus horikoshii.

At the initiation of chromosomal DNA replication, DNA primases synthesize short RNA primers, which are subsequently elongated by DNA polymerases. To understand the structural basis for the primer synthesis by archaeal/eukaryotic-type primases, the gene of the DNA primase from hyperthermophilic archaeon Pyrococcus horikoshii was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag at its amino terminus. The recombinant DNA primase was purified and crystallized by the hanging-drop vapor diffusion method at 293 K, with polyethylene glycol 8000 as the precipitant. The crystals belong to the P3(2)21 space group with unit-cell parameters a = b = 77.8, c = 129.6 A, and alpha = beta = 90 degrees, gamma = 120 degrees. Crystals of the selenomethionine derivative were obtained by means of a cross-seeding method using native crystals. The data for the native and selenomethionine-substituted crystals were collected to 1.8 and 2.2 A resolution, respectively, with synchrotron radiation at SPring-8 under flash-frozen conditions at 100 K. The four wavelength MAD data provided a phase to determine the structure of the primase at 2.2 A resolution.     

Purification, crystallization and preliminary crystallographic investigations of selenium-containing phycocyanin from selenium-rich algae (Spirulina platensis)

The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chromatographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P2₁ and cell parameters a =108.0 Å , b = 117.0 Å , c = 184.0 Å , β = 90.2° and 12(αβ ) units in the asymmetric unit was obtained by using (NH₄)₂SO₄ as precipitant. These crystals diffract up to 2.8 Å . Form II crystal obtained by using PEG4000 as precipitant belongs to space group P63 with unit cell constants a = 155.0 Å , c = 40.3 Å , γ =120.0° and one(αβ ) unit in the asymmetric unit. The crystals diffract beyond 2.9 Å . The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.     

Single crystals of bovine heart cytochrome c oxidase at fully oxidized resting, fully reduced and CO-bound fully reduced states are isomorphous with each other.

Fully reduced and CO-bound fully reduced forms of cytochrome c oxidase from beef heart muscle were crystallized in the presence of sodium ascorbate under N2 or CO atmosphere. Hexagonal bipyramidal and tetragonal crystals were obtained for both forms depending on buffer species. The hexagonal bipyramidal crystals, as large as 0.6 mm in the largest dimension, diffracted X-rays at 7 A resolution, showing an identical space group and cell dimension, P6(2) or P6(4) and a = b = 209 A, c = 283 A, respectively. These parameters coincide with those for crystals of the fully oxidized resting enzyme. This result suggests that a large conformational change, like a subunit arrangement, is not induced by the redox change and/or binding of CO (and possibly O2) to heme a3.