Crystallization and preliminary crystallographic analysis of ribonuclease H from Thermus thermophilus HB8

Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6 ₁22 (or P6 ₅22), with unit cell parameters a = b = 44.7 Å, c = 314.7 Å. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 Å resolution. © 1993 Wiley-Liss, Inc.     

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and Its complex with tRNAHis

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNA^His. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2₁2₁2 with cell parameters a = 171.3 Å, b = 214.7 Å, c = 49.3 Å, α = β = γ = 90°. A complete data set to a resolution of 2.7Å with an R_merge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNA_His shows that the crystals are tetragonal with cell parameters a = b = 232 Å, c = 559 Å, α = β = γ = 90° and diffract to about 4.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray studies of ornithine decarboxylase from Trypanosoma brucei

Trypanosoma brucei ornithine decarboxylase, expressed and purified from E. coli, has been crystallized by the vapor diffusion method using PEG 3350 as a precipitant. The crystals belong to the monoclinic space group P2₁ and have cell constants of a = 66.3 Å, b = 151.8 Å, c = 83.7 Å, and β = 101.2°. While larger crystals are twinned, smaller crystals (0.4 × 0.3 × 0.05 mm³) are single.     

Observation of a sterically unfavorable side-chain conformation in a leucyl residue: Crystal and molecular structure of L-leucyl–L-leucine · DMSO solvate

The crystal structure of a dipeptide L -leucyl–L -leucine (C₁₂H₂₄N₂O₃) has been determined. The crystals are monoclinic, space group P2₁, with a = 5.434(4) Å, b = 15.712(7) Å, c = 11.275(2) Å, β = 100.41(1)°, and Z = 2. The crystals contain one molecule of dimethyl sulfoxide (DMSO) as solvent of crystallization for each dipeptide molecule. The structure has been solved by direct methods and refined to a final R index of 0.059 for 920 reflections (sinθ/λ ? 0.60 Å^?1) with I ? 2σ (I). The trans peptide unit shows substantial degree of non-planarity (Δω = 14°). The peptide backbone adopts an extended conformation with torsion angles of ψ₁ = 138(1)°, ω₁ = 166(1)°, ?₂ = ? 149.3(7)°, ψ₂₁ = 164.2(7)°, and ψ₂₂ = ? 15(1)°. For the first leucyl residue, the side-chain conformation is specified by the torsion angles ¹χ₁ = 176.7(7)°, ¹χ₂₁ = 62(1)°, ¹χ₂₂ = ? 177.4(8)°; the second leucyl residue adopts a Sterically unfavorable conformation with ²χ₁ = 61(1)°, ²χ₂₁ = 97(1)°, and ²χ₂₂ = ?151(1)°. The packing involves head-to-tail interaction of peptide molecules and segregation of polar and nonpolar regions. The DMSO molecule is strongly hydrogen bonded to the terminal NH group. © 1994 John Wiley & Sons, Inc.     

Crystallization and preliminary X-ray diffraction data of two heparin-binding fragments of human fibronectin

Two different heparin-binding fragments of human fibronectin have been crystallized in forms which are suitable for crystal structure analyses. The 30 kDa hep-2A fragment, consisting of type III domains 12–14, was crystallized from solutions containing ammonium sulfate or polyethylene glycol 6000. The crystals grown in ammonium sulfate solutions were orthorhombic with space group I222 or I2₁2₁2₁ with a = 68.1 Å, b = 88.6 Å, and c = 144.9 Å. The crystals grown in polyethylene glycol solutions are hexagonal with space group P6₁22 or P6₅22 witha a = b = 66.7 Å and c = 245.7 Å. The 40 kDa hep-2B fragment, consisting of type III domains 12–15, was also crystallized from solutions containing ammonium sulfate with the addition of glycerol. Glycerol proved an effective agent for reducing the number of crystals in the crystallization experiments, and thus, increasing the size of the crystals in these experiments. This crystal form is nearly isomorphous to the orthorhombic form of the hep-2A fragment with space group I222 or I2₁2₁2₁ and a = 67.5 Å, b = 87.0 Å, and c = 144.3 Å. All crystal forms diffract to at least 3.5 Å resolution and contain a single molecule in the asymmetric unit. © 1993 Wiley-Liss, Inc.     

Molecular structure of the cis-syn photodimer of d(TpT) (cyanoethyl ester)

The three-dimensional structure was determined by x-ray crystallography for d(T[p](CE)T), a uv photoproduct of the cyanoethyl (CE) derivative of d(TpT), having the cis-syn cyclobutane (CB) geometry and the S-configuration at the chiral phosphorus atom. The crystals of C₂₃H₃₀N₅O₁₂P · 2H₂O belong to the orthorhombic space group P2₁2₁2₁ (Z = 4), with cell dimensions a = 11.596 Å, b = 14.834 Å, and c = 15.946 Å, containing two water molecules per asymmetric unit. The CB ring is puckered with a dihedral angle of 151°. The two pyrimidine bases are rotated by –29° from the position of direct overlap of their corresponding atoms. This represents a major distortion of DNA, since in DNA adjacent thymines are rotated by +36°. The pyrimidine rings are puckered with Cremer–Pople parameters for T[p] and in parentheses [p]T: Q: 0.24 Å (0.31 Å); θ: 123° (120°); ?: 141° (86°). These represent half-chairs designated as ⁶H₁ (T[p]) and ₆H⁵ ([p]T). The CB and pyrimidine ring conformations are interrelated, and we postulate that they execute a coupled interconversion in solution. The T[p] segment has the syn glycosyl conformation, a ²T₃ sugar pucker, and gauche^? conformation at C4′-C5′; the [p]T segment is anti, ³T₄, trans. The C5′-O5′ torsion of the [p]T unit is –124.5°, and the C3′-O3′ torsion of the T[p] unit is –152.9°. Bond angles and bond lengths involving the phosphorus atom are similar to those of other phosphotriesters. The P-O3′ and P-05′ torsion angles are –138.1° and 58.6°, respectively. Several intermolecular (but no intramolecular) hydrogen bonds are found in the crystal.     

Expression and crystallization of the yeast Hsp82 chaperone,and preliminary x-ray diffraction studies of the amino-terminal domain

Expression of the Saccharomyces cerevisiae Hsp82 chaperone in a pep4-3- and hsc82-deficient strain of S. cerevisiae yielded over 25% of the total cell protein as intact Hsp82. Similarly, the amino-terminal domain (residues 1–220) of Hsp82 was expressed to 18% of the total cell protein. Crystals of the intact Hsp82 were readily obtained. The crystals were very fragile, suggesting a high solvent content, and diffracted to approximately 8 Å. Tetragonal bipyrimidal crystals of the amino-terminal domain of Hsp82 were readily obtained under a variety of different conditions. The crystals have primitive tetragonal space group (P422, P4₁22, or its enantiomorph P4₃22) with unit cell dimensions of a = 75.1 Å and c = 111.3 Å, contain 60% by volume solvent, and diffract to 2.5 Å resolution. Addition of 25% glycerol to the mother liquor gave rise to large rod-shaped crystals. The crystals diffract to 2.8 Å resolution, have an orthorhombic space group (P222₁, P2₁2₁2, or P2₁2₁2₁) with cell dimensions of a = 45.2 Å, b = 115.4 Å, and c = 116.9 Å, and a solvent content of 58% by volume. © 1996 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of an a1/α2/DNA ternary complex

Crystals have been obtained of a ternary complex containing the yeast a1/α2 homeodomain heterodimer bound to a 21-base pair DNA site containing two 5′ overhanging bases at each end. The crystals are grown from cobaltic hexamine and form in space group P6₁ or P6₅ with a = b = 133 Å, c = 45.4 Å. Crystals that are flash-frozen at ?179°C diffract to 2.7 Å along the c-axis and to 2.4 Å in perpendicular directions. The crystals contain one protein–DNA complex in the crystallographic asymmetric unit. © 1995 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray diffraction analyses of the bacterial chemotaxis receptor modifying enzymes

Bacterial chemotaxis receptor modifying enzymes from Salmonella typhimurium have been crystallized using microseeding techniques. The crystals of the S-adenosyl-L -methionine-dependent methyl transferase, CheR, belong to the monoclinic space group P2₁ with cell constants a = 55.1 Å, b = 48.1 Å, c = 63.1 Å, β = 112.3°. The crystals of the catalytic domain of the methylesterase, CheB, belong to the trigonal space group P3₂21 or P3₁21 with unit cell dimensions of a = b = 63.4 Å, c = 86.8 Å. Both crystals contain one molecule per asymmetric unit and have calculated Matthews' volumes of 2.4 ų/Da. © 1995 Wiley-Liss, Inc.     

Crystallization of a scRIP—gelonin isolated from plant seeds Gelonium multiforum

Single crystals of the protein gelonin isolated from the seeds of Gelonium multiforum have been grown at room temperature by vapor diffusion method. The crystals are monclinic with a = 49.4 Å, b = 44.9 Å, c = 137.4 Å, and β = 98.3°. The space group is P2₁, with two molecules in the asymmetric unit which are related by a noncrystallographic 2-fold axis along ψ =13° and ? =88°. The crystals diffract X-rays to high resolution, making it possible to obtain an accurate structure of this single chain ribosome inactivating protein. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B

The Δ⁵-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of a = 36.48 Å, b = 74.30 Å, c = 96.02 Å, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Å resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.     

Crystallization and preliminary X-ray diffraction study of the green flavoenzyme 5-Hydroxyvaleryl-CoA dehydratase/dehydrogenase from Clostridium aminovalericum

The bifunctional flavoenzyme 5-hydroxyvaleryl-CoA dehydratase/ dehydrogenase has been crystallized from solutions containing ammonium sulfate (form I) or polyethylene glycol (form II) as precipitant. In both cases, the crystals grew in the monoclinic space group C2. The unit cell dimensions for form I crystals were determined as a = 162.8 Å, b = 71.8 Å, c = 83.5 Å, β = 109.1°. Corresponding values for form II crystals were a = 161.2 Å, b = 71.6 Å, c = 82.2 Å, β = 109.3°. In both cases most probably there are two monomers per asymmetric unit. The crystals diffract to about 2 Å resolution and are rather stable in the X-ray beam. © 1994 Wiley-Liss, Inc.     

A monoclonal antibody fab fragment crystallized with and without a peptide epitope from HIV

The Fab fragment of CB 4-1, a monoclonal murine antibody against HIV protein p24, has been produced. It forms a complex with a synthetic antigen, an epitope of p24 made up of 11 amino acids, with the binding constant k_d = 3.6 × 10^?9 M. Crystals of hexagonal and orthorhombic space group has been obtained by cocrystallization of the Fab with the epitope and crystallization without the epitope, respectively. In either case, the crystals are suitable for X-ray structural analysis. Crystals of the Fab fragment cocrystallized with the peptide have the space group P 6₃22 with cell dimensions of a = b = 105 Å, c = 297 Å. Fab crystals without the epitope are in space group C 222 with cell dimensions a = 110.1 Å, b = 110.2 c Å = 150.1 Å. © 1993 Wiley-Liss, Inc.     

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.     

Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl^? buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 Å. The crystals belong to the trigonal lattice type with the spacegroup P3₁21 or P3₂21, respectively, have the cell dimensions a = b = 96.8 Å, c = 196.2 Å, α = β = 90°,γ = 120°. The crystals have a theoretical packing density of 2.7 ų/Da, assuming one molecule with a molecular weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic study of human CksHs1: A cell cycle regulatory protein

The cell cycle regulatory protein CksHs1 has been crystallized in a form suitable for X-ray studies. CksHsl crystals were grown in the presence of vanadate, a phos-phatase inhibitor, but were also obtained with phosphate or tungstate as a cofactor. They belong to the hexagonal space group P6₁22 with unit cell dimensions: a=b=94 Å, c=131.6 Å, and γ =120. The crystals grown in the presence of vanadate diffract X-rays to at least 2.8 Å. Molecular replacement results from the homologous human CksHs2 structure reveal that a dimer forms the crystal habit, giving the unusual V_m value of 4.4 ų/Da or a solvent content of 72%. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of ImmE7 protein of colicin E7

The ImmE7 protein, which can bind specifically to the DNase colicin E7 and neutralize its bactericidal activity, has been purified and crystallized in two different crystal forms by vapor diffusion method. The orthorhombic crystals belong to space group I222 or I2₁2₁2₁ and have unit cell dimensions a = 75.1 Å, b = 50.5 Å, and c = 45.4 Å. The second form is monoclinic space group P2₁ with ceil dimensions a = 29.3 Å, b = 102.7 Å, c = 53.0 Å and β = 91.5°. The orthorhombic crystals diffract to 1.8 Å resolution, and are suitable for high-resolution X-ray analysis. © 1995 Wiley-Liss, Inc.     

Conformation of the flexible bent helix of Leu1-zervamicin in crystal C and a possible gating action for ion passage

The membrane channel-forming polypeptide, Leu¹-zervamicin, Ac-Leu-Ile-Gln-Iva-Ile⁵-Thr-Aib-Leu-Aib-Hyp¹⁰-Gln-Aib-Hyp-Aib-Pro¹⁵-Phol (Aib: α-aminoisobutyric acid; Iva: isovaline; Hyp: 4-hydroxyproline; Phol: phenylalininol) has been analyzed by x-ray diffraction in a third crystal form. Although the bent helix is quite similar to the conformations found in crystals A and B, the amount of bending is more severe with a bending angle ≈ 47°. The water channel formed by the convex polar faces of neighboring helices is larger at the mouth than in crystals A and B, and the water sites have become disordered. The channel is interrupted in the middle by a hydrogen bond between the OH of Hyp (10) and the NH₂ of the Gln (11) of a neighboring molecule. The side chain of Gln (11) is wrapped around the helix backbone in an unusual fashion in order that it can augment the polar side of the helix. In the present crystal C there appears to be an additional conformation for the Gln (11) side chain (with ≈ 20% occupancy) that opens the channel for possible ion passage. Structure parameters for C₈₅H₁₄₀N₁₈O₂₂ · xH₂O · C₂H₅OH are space group P2₁2₁2₁, a = 10.337(2) Å, b = 28.387(7) Å, c = 39.864(11) Å, Z = 4, agreement factor R = 12.99% for 3250 data observed > 3σ(F), resolution = 1.2 Å. © 1994 John Wiley & Sons, Inc.     

Crystallization of the bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase domain of the human trifunctional enzyme

Methylenetetrahydrofolate([H₄] folate) dehydrogenase (D) and methenyl[H₄] folate cyclohydrolase (C) coexist as a bifunctional enzyme (DC) or as the amino-terminal domain of a trifunctional enzyme (DCS) where the third activity is 10-formyl[H₄]lfolate synthetase (S). Two crystal forms of the DC domain of the human cytosolic DCS enzyme have been grown from polyethyleneglycol solution. The monoclinic P2₁ crystals diffract to 2.8 Å with a = 72.5 Å, b = 68.5 Å, c = 125.2 Å, and β = 91.8° but were found to be twinned. The orthorhombic P2₁2₁2₁ crystals diffract to 2.5 Å with a = 67.7 Å, b = 135.9 Å, c = 61.6 Å, and contain two molecules per asymmetric unit. Proteins 26:479–480 © 1996 Wiley-Liss, Inc.     

α,β-Dehydro-amino acid residues in the design of peptide structures: Synthesis,crystal structure,and molecular conformation of two homologous peptides—N-Ac-Dehydro-Phe-L-Leu-OCH3 and N-Ac-Dehydro-Phe-NorVal-OCH3

The dehydro-residue containing peptides N-Ac-dehydro-Phe-L -Leu-OCH₃ ( I ) and N-Ac-dehydro-Phe-NorVal-OCH₃ ( II ) were synthesized by the usual workup procedures. The peptides crystallize from their solutions in methanol in space group P6₅: ( I ) a = b = 12.528(2) Å, c = 21.653(5) Å; ( II ) a = b = 12.532(2) Å, c = 21.695(4) Å. The structures were determined by direct methods. Both peptides adopt similar conformations with ?,ψ of dehydro-Phe as follows: ( I ) ?57.0(5)° and ?37.0(5)°; ( II ) ?56.0(5),° and ?37.5(5)°. The observed data on dehydro-Phe when placed at the (i + 1) position show that the ?,ψ values of dehydro-Phe are either ?60°, 140° or ?60°, ?30°. The conformation of ?60°, 140° can be accommodated only with a flexible residue at the (i + 2) position while the ?,ψ values of ?60°, ?30° are obtained with a bulky residue at the (i + 2) position as in the present structures. The molecules are packed in a helical way along the c axis. These are held by two strong intermolecular hydrogen bonds involving both NH as donors and acetyl group and dehydro-Phe oxygen atoms as acceptors. © 1994 John Wiley & Sons, Inc.