Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase.

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.     

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.     

Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein.

The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.     

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.     

Isolation, characterization, and preliminary X-ray diffraction data for a serine protease from Penicillium cyclopium

The major extracellular protein of Penicillium cyclopium has been isolated from its culture media and purified by ammonium sulfate fractionation, gel, and ion-exchange chromatography. We show this secreted protein to be endopeptidase. The molecular weight is approximately 32,000, the pI is 5.0, and the pH optimum using a variety of protein and synthetic substrates is around 7.0. Inhibition studies show that the protease is not inhibited by pepstatin nor by p-chloromercuribenzoic acid, indicating, respectively, that it is not an aspartyl protease nor a thiol protease. Complete inhibition is observed, however, with phenylmethanesulfonyl fluoride. Three crystal forms suitable for high resolution x-ray diffraction studies have been obtained from this purified protease with reflections being observed to well beyond 3.0 A resolution. One form having a needle morphology is of the orthorhombic crystal class and has space group P2(1)2(1)2(1). The unit cell dimensions are a = 41.9 A, b = 43.2 A, and c = 111.5 A with 1 molecule of the protease occurring in the asymmetric unit. The second form grown at pH values less than 6.0 has a plate morphology, is of orthorhombic space group P2(1)2(1)2(1), and has unit cell dimensions a = 59.12 A, b = 62.33 A, and c = 70.62 A. The third form is polyhedral in habit, is also of space group P2(1)2(1)2(1), and appears when the pH of the mother liquor is greater than 7.0. The cell dimensions of this crystal form are a = 57.07 A, b = 58.82 A, c = 70.79 A, and again there is 1 molecule/asymmetric unit. Three-dimensional structural analysis by x-ray diffraction is now underway. All crystal forms are somewhat denser than the norm having mass to volume ratios of 1.58, 2.00, and 1.85 A3/dalton, respectively.     

Preliminary crystallographic analysis of 5-carboxymethyl-2-hydroxymuconate isomerase from Escherichia coli

Escherichia coli 5-carboxymethyl-2-hydroxymuconate (CHM) isomerase was purified from an overexpressing cell line. The enzyme has been crystallized from ammonium sulphate in two different crystal forms. One of these has been analysed and found to be orthorhombic I222 or I2(1)2(1)2(1) with cell dimensions a = 88 A, b = 89 A, c = 121 A. The asymmetric unit contains two dimers (Vm = 2.11 A3/dalton). The crystals diffract to beyond 3.0 A resolution and are stable to irradiation with X-rays. Data have been collected to 3.0 A resolution and a search for potential heavy-metal derivatives is in progress.     

Structure of the extended-spectrum class C beta-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion.

A class C beta-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improved hydrolytic activity for oxyimino beta-lactam antibiotics has been analyzed by X-ray crystallography to 1.8 A resolution. Relative to the wild-type P99 beta-lactamase, this natural mutant contains a highly unique tandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4 kDa chromosomal beta-lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate in space group P2(1)2(1)2 with cell dimensions a = 78.0 A, b = 69.5 A, and c = 63.1 A. The crystal structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20 for all nonzero data from 8 to 1.8 A. Deviations of model bonds and angles from ideal values are 0.008 A and 1.4 degrees, respectively. Overlay of alpha-carbon atoms in the GC1 and P99 beta-lactamases results in an rms deviation of 0.6 A. Largest deviations occur in a loop containing Gln120 and in the Omega loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width of the opening to the substrate binding cavity, as measured from the 318-324 beta-strand to two loops containing Gln120 and Tyr150 on the other side, is 0.6-1.4 A wider than in P99. It is suggested that conformational flexibility in the expanded Omega loop, and its influence on adjacent protein structure, may facilitate hydrolysis of oxyimino beta-lactams by making the acyl intermediate more open to attack by water. Nevertheless, backbone atoms in core catalytic site residues Ser64, Lys67, Tyr150, Asn152, Lys318, and Ser321 deviate only 0.4 A (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 at pH 8, is consistent with the requirement for a lower than normal pK(a) for this residue.     

Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution.

The crystal structure of beta-lactamase TEM1 from E. coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S. Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme. However, global superposition of the two proteins shows significant differences.     

Crystallization and characterization of the high molecular weight form of nerve growth factor (7 S NGF)

A high molecular weight form of nerve growth factor (7 S NGF) has been crystallized in two crystal forms from polyethylene glycol 4000 by the vapour diffusion technique. The orthorhombic form A belongs to the space group P2(1)2(1)2(1) and has cell dimensions of a = 95.6, b = 96.5 and c = 147.0 A. With synchrotron X-ray radiation, these crystals diffract to 2.8 A resolution. They contain an intact 7 S NGF complex in the asymmetric unit. The tetragonal form B, which grows at similar conditions to the A form, belongs to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = 97.4, b = 97.4 and c = 308.3 A. These crystals diffract to 3.6 A resolution and contain one 7 S complex per asymmetric unit. Native X-ray data have been collected to 3.3 A for the A form and to 5.0 A for the B form, both using synchrotron radiation.     

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.     

Crystal structure of a light-harvesting protein C-phycocyanin from Spirulina platensis

The crystal structure of C-phycocyanin, a light-harvesting phycobiliprotein from cyanobacteria (blue-green algae) Spirulina platensis has been solved by molecular replacement technique. The crystals belong to space group P2(1) with cell parameters a = 107.20, b = 115.40, c = 183.04 A; beta = 90.2 degrees. The structure has been refined to a crystallographic R factor of 19.2% (R(free) = 23.9%) using the X-ray diffraction data extending up to 2.2 A resolution. The asymmetric unit of the crystal cell consists of two (alphabeta)6-hexamers, each hexamer being the functional unit in the native antenna rod of cyanobacteria. The molecular structure resembles that of other reported C-phycocyanins. However, the unique form of aggregation of two (alphabeta)6-hexamers in the crystal asymmetric unit, suggests additional pathways of energy transfer in lateral direction between the adjacent hexamers involving beta155 phycocyanobilin chromophores.     

Expression, purification, characterization and crystallization of flap endonuclease-1 from Methanococcus jannaschii

A gene coding for a protein homologous to the flap endonuclease-1 (FEN-1) was cloned from Methanococcus jannaschii, overexpressed, purified and characterized. The gene product from M. jannaschii shows 5' endo-/exonuclease and 5' pseudo-Y-endonuclease activities as observed in the FEN-1 in eukaryotes. In addition, Methanococcus jannaschii FEN-1 functions effectively at high concentrations of salt, unlike eukaryotic FEN-1. We have crystallized Methanococcus jannaschii FEN-1 and analyzed its preliminary character. The crystal belongs to the space group of P2(1) with unit cell dimensions of a = 58.93 A, b = 42.53 A, c = 62.62 A and beta = 92.250. A complete data set has been collected at 2.0 A resolution using a frozen crystal.     

Purification and crystallization of the EcoRV restriction endonuclease

The type II restriction endonuclease EcoRV purified from a genetically engineered, overproducing strain has been crystallized. Four crystal forms all obtained by precipitation with polyethylene glycol 4000 have been characterized. Two of these are suitable for high resolution structure analysis. Both are orthorhombic, have space group P2(1)2(1)2(1) and have similar unit cell dimensions of a = 58.2 A, b = 71.7 A, c = 130.6 A (form A) and a = 59.9 A, b = 74.5 A, c = 121.8 A (form B). They diffract to about 2A resolution and appear to have one dimer of 2 X 29,000 daltons in the asymmetric unit.     

Crystallization and preliminary X-ray studies of the liganded lysine, arginine, ornithine-binding protein from Salmonella typhimurium.

The periplasmic binding protein LAO from Salmonella typhimurium, which is involved in lysine, arginine and ornithine transport, has been crystallized together with one of its ligands, arginine (LAO-Arg). Preliminary X-ray diffraction studies of LAO-Arg crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1) and has the unit cell dimensions of a = 37.65 A, b = 59.45 A, c = 115.91 A. Crystals of the LAO-Arg complex diffract beyond 2.0 A resolution.     

Crystallization and preliminary X-Ray diffraction analysis of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp. GK16

Glutaryl-7-aminocephalosporanicacid acylase from Pseudomonas sp. GK16 produces glutaryl-7-aminocephalosporanic acid, a key intermediate for the synthesis of cephem antibiotics. Sequence alignment suggests that the enzyme may belong to the N-terminal nucleophile hydrolase superfamily including penicillin G acylase. The enzyme is an (alphabeta)(2) heterotetramer of two nonidentical subunits. These subunits are derived from a nascent precursor polypeptide that is cleaved proteolytically through a two-step autocatalytic process upon folding. The enzyme has been crystallized using the vapor diffusion method. A bipyramidal crystal form was obtained from a solution containing polyethylene glycol (MW 3350) and calcium chloride. Complete diffraction data sets have been collected up to 2.8 A resolution. The crystal is tetragonal with the space group P4(1)2(1)2 or P4(3)2(1)2 and the unit cell parameters are a = b = 73.5 A, c = 380.3 A. Considerations of the possible values of V(m) account for the presence of a tetramer in the asymmetric unit.     

Crystallization and preliminary X-ray studies on Sulfolobus acidocaldarius ferredoxin.

Ferredoxin from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius, has been crystallized. The space group is P4(3)2(1)2 or P4(1)2(1)2 and the cell dimensions are a = b = 50.12 A and c = 69.52 A. The Vm value is calculated to be 1.88 A3/Da, assuming one molecule per asymmetric unit. The crystal diffracts X-rays beyond 2.0 A resolution.     

Crystallographic study of endogenous alpha-amylase inhibitor from wheat

Endogenous alpha-amylase inhibitor from wheat has been crystallized by a microdialysis method. There are two forms of monoclinic crystal in a microdialysis cell with a space group of P2(1). The unit cell dimensions are a = 43.5 A, b = 64.8 A, c = 32.2 A, beta = 113 degrees for the rod-like crystal, and a = 42.5 A, b = 65.2 A, c = 32.2 A, beta = 112 degrees for the plate-like crystal. The former is suitable for structure analysis because it gives the sharp diffraction beyond 2.0 A resolution, and the latter tends to form a twin crystal. A heavy-atom derivative has been successfully prepared with the heavy-atom reagent K2PtCl4, and structure analysis is in progress.     

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.     

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.     

New crystal form of cytosolic chicken aspartate aminotransferase suitable for high-resolution X-ray analysis

Four new crystal forms of chicken cytosolic aspartate aminotransferase have been grown from polyethylene glycol solutions. Crystals of the unliganded enzyme and of enzyme liganded with maleate diffract to 1.8 A resolution. Both the free and maleate-liganded enzymes crystallize in space group P2(1)2(1)2(1), but display slightly different cell dimensions (a = 56.9 A, b = 126.9 A and c = 124.6 A versus a = 56.5 A, b = 126.1 A and c = 124.6 A). The influence of various divalent metal ions, dioxane and non-ionic detergent beta-octylglucoside on crystallization has been investigated. The best crystals of liganded enzyme were obtained in the presence of Mg2+ ions, and these crystals were used for data collection to 1.9 A resolution.