Two-dimensional crystallization of reaction centers from Chloroflexus aurantiacus

Two-dimensional crystals of photosynthetic reaction centers from Chloroflexus aurantiacus were obtained from protein-lipid-detergent micelles by detergent dialysis. The size of crystals was up to 2 microns. Some of them were multilayered crystals. However, other crystal forms were also observed. Preliminary image processing analysis showed that crystals of one crystal form referred to two-sided plane group p2 and had the following unit cell parameters: a = 17.6 nm, b = 18.0 nm, gamma = 84 degrees. The contour map of the crystal stain-excluding region was calculated by the Fourier-filtering procedure at about 2 nm resolution.     

Crystallization and preliminary X-ray diffraction analysis of staphylococcal enterotoxin type C.

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 A, respectively. The triclinic crystals have unit cell parameters a = 38.5 A, b = 43.7 A, c = 36.9 A, and interaxial angles alpha = 99.9 degrees, beta = 95.8 degrees, and gamma = 98.5 degrees. The tetragonal crystals are of space group P4(1)22 with unit cell parameters a = 43.4 A and c = 278.0 A.     

Crystals of P2 myelin protein in lipid-bound form

The P2 protein of peripheral nervous system myelin induces experimental allergic neuritis in rats, a model of Guillain-Barré syndrome in humans. Previous purification procedures have used acid extraction to obtain the protein in lipid-free form (LF-P2). Here, we have purified the P2 protein in lipid-bound form (LB-P2) by extracting myelin with the detergent CHAPS, followed by Cu(2+)-affinity column chromatography. All myelin lipids were present in the preparation as shown by high-performance thin-layer chromatography and mass spectrometry. The LB-P2 preparation, which differs from LF-P2 in solubility and in the secondary-structure composition, was dialyzed to remove unbound lipids and excess detergent and crystallized using the hanging-drop vapor diffusion technique. Crystals of lipid-bound P2 appeared usually very reproducibly within 2 weeks at pH 5.7 in polyethylene glycol 6000 (PEG6000) at concentrations of 20-30% (w/v), and larger crystals were obtained by additional sitting-drop crystallization. X-ray diffraction showed reflections up to 2.7A. The crystallization conditions (25-30% PEG6000, pH 5.0) and the unit cell dimensions (a = 94.5A, b = 94.5A, c=74.2A, alpha = beta = 90 degrees, gamma = 120 degrees ) of LB-P2 were different from those earlier described for LF-P2 (10% PEG4000, pH 3, and unit cell dimensions a = 91.8A, b = 99.5A, c = 56.5A, alpha = beta = gamma = 90.0 degrees ). It is important that P2 has been crystallized with specifically bound lipids; therefore, solving this new crystal structure will reveal details of this protein's behavior and role in the myelin sheath.     

Crystallization and preliminary x-ray diffraction studies of recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta has been crystallized into a tetragonal cell. The unit cell constants are a = b = 54.9 A, c = 76.8 A, and alpha = beta = gamma = 90 degrees. The crystals diffract to better than 1.9 A and are suitable for high resolution data collection. The crystallization conditions and general crystal data are presented.     

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.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.     

Preliminary crystallographic study of bluetongue virus capsid protein, VP7.

Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.     

Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein.

Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.     

Crystallization of chicken egg white cystatin, a low molecular weight protein inhibitor of cysteine proteinases, and preliminary X-ray diffraction data

The shorter-chain form of chicken egg white cystatin has been crystallized in 1.6 M-phosphate buffer at pH 4.0 by vapour diffusion. The crystals are of trigonal space group P3121 (or P3221), have cell constants a = b = 47.9 A, c = 87.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and contain one molecule of 12,191 molecular weight per asymmetric unit. They diffract well to about 2.0 A resolution and are suitable for X-ray crystal structure analysis.     

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.     

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.     

The lumazine synthase-riboflavin synthase complex of Bacillus subtilis. Crystallization of reconstituted icosahedral beta-subunit capsids

The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits. The enzyme has been purified from the derepressed mutant H 94 of B. subtilis by a novel efficient procedure using column chromatography and preparative crystallization. Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0. Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications. A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees. The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution. A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees. These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60). A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees. The space group of this modification could not be determined unambiguously.     

Preliminary X-ray analysis of crystals of plasminogen activator inhibitor-1

Crystals of bacterially expressed plasminogen activator inhibitor (PAI-1) suitable for X-ray diffraction analysis have been obtained from 8% (w/v) PEG 1500, pH 8.25. The space group is P1, and the lattice constants are a = 82.17 A, b = 47.82 A, c = 62.89 A, alpha = 90.00 degrees, beta = 106.90 degrees, gamma = 106.84 degrees. The diffraction limit is 2.3 A, and the unit cell contains two molecules of PAI-1. The crystals contain latent PAI-1 which can be partly reactivated by exposure to denaturants.     

Structural studies of a novel germination protease from spores of Bacillus megaterium

The amino acid sequence-specific protease (termed GPR) in the bacterium Bacillus megaterium initiates the rapid degradation of small, acid-soluble spore proteins during the germination of spores of this organism. GPR is synthesized during spore formation as an inactive zymogen termed P46, which later autoprocesses to a smaller active form termed P41, which acts during spore germination. However, GPR exhibits no obvious mechanistic or amino acid sequence similarity to any of the known classes of proteases. To initiate the determination of the mechanisms of P46 to P41 conversion, P46 inactivity, and P41 catalysis, B. megaterium GPR has been overexpressed in Escherichia coli and purified to homogeneity by anion-exchange and size exclusion chromatography, and crystals of both P46 and P41 have been obtained by the vapor diffusion method. P46 crystals diffracted x rays to 3.5 A but the crystals of P41 diffracted x rays to only 6.5 A. A native x-ray diffraction data set of P46 has been collected; the unit cell parameters are a = b = 76.8, c = 313.1 A, alpha = beta = gamma = 90 degrees; the space group is tetragonal P41212 or P43212. The asymmetric unit contains two monomeric molecules with a crystal volume per unit protein mass of 2. 85 A3/Da and a solvent content of about 57%. An isomorphous heavy atom derivative data set has also been obtained for P46 crystals with potassium dicyanoaurate (I).     

A crystallographic investigation on NADPH-adrenodoxin oxidoreductase

Single crystals of NADPH-adrenodoxin oxidoreductase were grown in 50 mM potassium phosphate (pH 7.4) containing 5% glycerol and ammonium sulfate. The crystals are monoclinic, belong to space group P21 and have dimensions of a= 83.4 A, b = 62.6 A, c = 59.3 A, alpha = gamma = 90 degrees, and beta = 107.1 degrees. There is one molecule per asymmetric unit.     

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 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.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Crystallization and preliminary X-ray studies of the Ia component of Clostridium perfringens iota toxin complexed with NADPH.

A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent. The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ). The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit. Native data were collected at 2.4-A resolution. The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference. The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit.     

Human liver cathepsin D. Purification, crystallization and preliminary X-ray diffraction analysis of a lysosomal enzyme.

The two-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, has been isolated from human liver. Isoelectric focusing revealed two major species of enzyme that differed by approximately 0.2 pI unit. Crystals suitable for X-ray diffraction analysis were prepared from acidic solutions using precipitation with ammonium sulfate. The hexagonal crystals diffracted X-rays to beyond 3.1 A resolution and belonged to space group P6(1) (or P6(5)) with cell constants a = b = 125.9 A, c = 104.1 A, gamma = 120.0 degrees. The crystals likely contain two molecules in the asymmetric unit, giving a solvent content of 56% (v/w). Biochemical analysis of crystals indicated that both isoforms were present in approximately equimolar proportions. Full structure determination of the enzyme is underway.