Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.     

Crystallization and preliminary structural studies of neurotrophin-3.

Neurotrophin-3 (NT-3) has been crystallized in 2 forms. Orthorhombic crystals, space group P2(1)2(1)2, diffracted to 2.8 A and have cell dimensions a = 39.1 A, b = 54.0 A, and c = 65.5 A. The second form is space group P4(3)2(1)2, with cell dimensions a = b = 67.1 A, and c = 107.9 A. The tetragonal crystals diffract to 2.8 A at room temperature and 2.5 A at -100 degrees C. The unit cell dimensions change significantly upon freezing, a = b = 66.1 A, and c = 102.8 A. Phases for the orthorhombic form were obtained by molecular replacement using nerve growth factor as the search model. A partially refined model of the NT-3 dimer (75% complete) was then oriented and positioned in the tetragonal cell.     

Purification and crystallization of thymidine kinase from herpes simplex virus type 1

Thymidine kinase from herpes simplex virus type 1 (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21) has been purified from an overexpression system and crystallized against ammonium sulfate by using the hanging-drop technique. The tetragonal crystals are of space group P4122 or P4322, and have unit cell dimensions a = b = 84 A, c = 180 A.     

Crystallization and preliminary X-ray diffraction data of two crystal forms of bovine heart creatine kinase

Two crystal forms of bovine heart creatine kinase, which are suitable for X-ray diffraction studies, have been grown at room temperature using 2-methyl-2,4-pentanediol as the precipitant at pH 7.2. The space group of the orthorhombic form is P2(1)2(1)2, with unit cell dimensions a = 133 A, b = 128 A and c = 65 A, and there is one dimeric molecule in the asymmetric unit. The space group of the tetragonal form is P4(2)2(1)2, with unit cell dimensions a = b = 132 A and c = 75 A, with one subunit in the asymmetric unit. The tetragonal crystals diffract to at least 2.0 A resolution.     

Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB.

The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells. CspB was crystallized in two different forms using vapor diffusion methods. The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A. The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A. These crystals grow with polyethylene glycol 4000 as precipitant.     

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.     

Proteins of the Bacillus stearothermophilus ribosome. Crystallization of proteins L30 and S5

Proteins L30 and S5 from the 50 S and 30 S subunits, respectively, of the Bacillus stearothermophilus ribosome have been crystallized. L30 crystals are tetragonal and the space group is P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 46.3 A and c = 61.4 A. S5 crystals are trigonal with the space group P3(1)21 (or P3(2)21) and cell dimensions a = b = 59.3 A and c = 109.8 A. In both cases, there appears to be a single molecule in the asymmetric unit.     

Crystallization and preliminary x-ray studies of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with activator and a transition state analogue

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been purified from spinach and crystallized by equilibrium vapor diffusion with polyethylene glycol 6000 as a precipitant. Crystals suitable for x-ray studies were obtained from a binary complex with a transition state analogue, 2-C-carboxy-D-arabinitol 1,5-bisphosphate, and a quaternary complex with 2-C-carboxy-D-arabinitol 1,5-bisphosphate, Mg2+, and HCO-3. Two forms of crystals were obtained in the presence of 2-C-carboxy-D-arabinitol 1,5-bisphosphate. Form B crystals are plates which have orthorhombic space group P2(1)2(1)2 with unit cell dimensions a = 184 A, b = 218 A, and c = 119 A. Form C crystals are tetragonal needles with space group I422 and with cell dimensions a = b = 275 A and c = 178 A. In both forms, the asymmetric unit contains half a molecule.     

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 crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.     

Crystallization and preliminary X-ray analysis of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase from rat liver has been crystallized from polyethylene glycol 4500 with sodium formate. The crystals are tetragonal rods and belong to space group P4(1)22 or P4(3)22 with unit cell dimensions a = b = 57.6 A and c = 304.1 A. They diffract to at least 2.4 A resolution and have one molecule per asymmetric unit.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Crystallization and preliminary x-ray diffraction studies of recombinant rabbit interferon-gamma

Two different crystal forms of recombinant rabbit IFN-gamma were obtained under different crystallization conditions. The first, a tetragonal form with space group P43212 or P41212, was obtained through vapor phase equilibration using the sitting drop rods technique with ammonium citrate as the major precipitating agent. The unit cell dimensions of this crystal form are a = b = 82.1 A and C = 116.3 A. These crystals diffract to 2.8 A resolution and contain a dimer in the asymmetric unit. A second crystal form was obtained by the batch method at pH 8.0 using sodium chloride as the precipitating agent. The crystals are hexagonal, space group P6122 or P6522, and with unit cell dimensions of a = b = 58.0 A and c = 169 A. This form contains monomer in the asymmetric unit and diffracts to greater than 2.7 A resolution. Both forms appear to be eminently suitable for further analyses and crystal structure solution.     

Crystallization and preliminary X-ray data for the exocellular beta-lactamase of Bacillus licheniformis 749/C

The exocellular beta-lactamase from Bacillus licheniformis 749/C has been crystallized from polyethylene glycol solution at pH 5.5. An X-ray examination of the monoclinic crystals shows the space group is P21, with unit cell dimensions a = 66.77 A, b = 93.77 A, c = 43.57 A and beta = 104.5 degrees. The asymmetric unit consists of two molecules of 28,500 Mr each. The crystals are suitable for structure analysis to at least 2 A resolution.     

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.     

Crystallization, activity assay and preliminary X-ray diffraction analysis of the uncleaved form of the serpin antichymotrypsin.

Crystals of recombinant wild-type antichymotrypsin have been prepared by the method of vapor diffusion with polyethylene glycol 4000 as a precipitant at pH 5.7. Two crystal forms are observed. One form belongs to tetragonal space group P4(3)2(1)2 (or P4(1)2(1)2) and has unit cell dimensions a = b = 126 A, c = 243 A, with two molecules in the asymmetric unit. The other crystal form belongs to orthorhombic space group P2(1)2(1)2(1) and has unit cell parameters of a = 73 A, b = 78 A and c = 80 A, with one molecular in the asymmetric unit. Diffraction intensity measurements have been made on the tetragonal crystal form to a limiting resolution of 4.1 A, and reflections have been observed on X-ray still photographs to a limiting resolution of 2.5 A for the orthorhombic form. An activity assay of redissolved tetragonal form crystals indicates that the uncleaved, functional serpin has been crystallized.     

Crystallization and preliminary X-ray analysis of gamma-aminobutyric acid transaminase

gamma-Aminobutyric acid transaminase from pig liver, an alpha 2 dimeric enzyme of Mr 110,100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P2(1), unit cell dimensions a = 82.1 A, b = 230.0 A, c = 70.3 A, beta = 123.9 degrees and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.     

Crystallization and preliminary X-ray studies of an aspartate aminotransferase mutant from Escherichia coli

Mutant aspartate aminotransferase V39L (Val39 replaced by Leu) from Escherichia coli has been crystallized into a monoclinic cell from a polyethylene glycol solution (pH 7.5) by vapor diffusion. The space group and the unit cell dimensions have been determined using a precession camera, a CAD4 diffractometer and a Nicolet Xentronics area detector to be P2(1) with a = 86.8 A, b = 79.9 A, c = 89.4 A, beta = 118.74 degrees. The crystals diffract to better than 2.3 A and are suitable for X-ray structure analysis.     

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.