Crystal structure of an open conformation of citrate synthase from chicken heart at 2.8-A resolution

The X-ray structure of a new crystal form of chicken heart muscle citrate synthase, grown from solutions containing citrate and coenzyme A or L-malate and acetyl coenzyme A, has been determined by molecular replacement at 2.8-A resolution. The space group is P4(3) with a = 58.9 A and c = 259.2 A and contains an entire dimer of molecular weight 100,000 in the asymmetric unit. Both "closed" conformation chicken heart and "open" conformation pig heart citrate synthase models (Brookhaven Protein Data Bank designations 3CTS and 1CTS) were used in the molecular replacement solution, with crystallographic refinement being initiated with the latter. The conventional crystallographic R factor of the final refined model is 19.6% for the data between 6- and 2.8-A resolution. The model has an rms deviation from ideal values of 0.034 A for bond lengths and of 3.6 degrees for bond angles. The conformation of the enzyme is essentially identical with that of a previously determined "open" form of pig heart muscle citrate synthase which crystallizes in a different space group, with one monomer in the asymmetric unit, from either phosphate or citrate solution. The crystalline environment of each subunit of the chicken enzyme is different, yet the conformation is the same in each. The open conformation is therefore not an artifact of crystal packing or crystallization conditions and is not species dependent. Both "open" and "closed" crystal forms of the chicken heart enzyme grow from the same drop, showing that both conformations of the enzyme are present at equilibrium in solution containing reaction products or substrate analogues.     

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

Molecular orientation and position of the pig M4 and H4 isoenzymes of lactate dehydrogenase in their crystal cells

Ternary complexes of M₄ and H₄ isoenzymes of porcine lactate dehydrogenase have been crystallized, the M₄ isoenzyme in space group P22₁2₁ with one half molecule per asymmetric unit, and the H₄ isoenzyme in space group C₂ with one whole molecule per asymmetric unit. The orientation and position of the tetramers in their unit cells have been determined by X-ray analysis. Rotation function results comparing the ternary complexes of the pig M₄ isoenzyme with the known structure of the dogfish M₄ enzyme not only defined the direction but also permitted recognition of the individual P, Q and R molecular 2-fold axes. The position of the molecular center was determined by placing a properly oriented dogfish M₄ lactate dehydrogenase electron density into the pig muscle cell. Structure factors were calculated as the molecular center was varied along the common crystallographic and molecular 2-fold axis and compared with observed amplitudes. Precession photographs of the three major zones of the monoclinic pig H₄ isoenzyme exhibited striking similarities to the corresponding zones of the orthorhombio pig M₄ isoenzyme, in spite of the differences in space groups. These similarities permit the determination of approximate phases from the implied orientation and position of the pig H₄ lactate dehydrogenase molecule in its monoclinic cell.     

Preliminary X-ray studies on an unspecific NAD(P)H dehydrogenase from human erythrocytes

Crystals of a relatively unspecific NAD(P)H dehydrogenase from human erythrocytes suitable for X-ray analysis have been grown. They belong to space group P4₁2₁2 or its enantiomorph P4₃2₁2 with unit cell dimensions: $\text{a = b = 79.3}\text{A}\text{?}\text{and}\text{c = 38.1}\text{A}\text{?}$. The asymmetric unit contains one molecule of M_r 18,000.     

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 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.     

Crystallization and preliminary X-ray analysis of pig kidney DOPA decarboxylase.

DOPA decarboxylase from pig kidney, an alpha 2 dimeric enzyme of Mr = 107,000, has been crystallized by the vapour diffusion method with ammonium sulphate as precipitant. The crystals belong to the space group P6(2) (or its enantiomer P6(4)) and have unit cell dimensions of a = b = 155.9 A, c = 87.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. They diffract to 2.6 A resolution. There is one dimeric molecule per asymmetric unit. Rotation function studies have revealed the orientation of the non-crystallographic 2-fold axis of the dimer in the asymmetric unit.     

Purification,crystallization, and preliminary X-ray studies on the rhizome lectin from stinging nettle and its complex with NN′N″-triacetylchitotriose

Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN′N″ -triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P2₁2₁2₁ with unit cell dimensions a = 54.3 (1) Å, b = 62.2 (1) Å, and c = 92.4 (2) Å, and diffracts to 3.0 Å resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P2₁2₁2₁ with cell constants a = 37.69 (4) Å, b = 48.97 (6) Å, and c = 57.32 (4) Å. These crystals diffract to at least 2.0 Å resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way. © 1993 Wiley-Liss, Inc.     

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.     

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 X-Ray Diffraction Analysis of Group 5 Mite Allergen fromDermatophagoides pteronyssinus

Crystals of the 14-kDa group 5 allergen fromDermatophagoides pteronyssinus(Der p 5) have been obtained at low pH and diffract to 3-Å resolution using a conventional x-ray source. The crystals belong to tetragonal space group P4₁2₁2 or P4₃2₁2, with unit cell parametersa=b= 114 Å andc= 234 Å. A self-rotation search revealed a 432 point symmetry and thus suggested 96 molecules in one unit cell, hence 12 monomers in each asymmetric unit.     

Preliminary crystallographic data for adenylosuccinate synthetase from rabbit skeletal muscle

Single crystals of adenylosuccinate synthetase, an essential component of purine biosynthesis, extracted from rabbit skeletal muscle were prepared as suitable specimens for X-ray structure analysis. The crystal belongs to the space group P4₃2₁2 or P4₁2₁2 with unit cell dimensions $\text{a = b = 71.2}\text{A}\text{?}\text{, c = 194.8}\text{A}\text{?}$. The asymmetric unit contains one protein molecule of 54,000 molecular weight.     

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage φ29 connector particle

The connector or portal particle from double-stranded DNA bacteriophage φ29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a=416.86 Å, b=227.62 Å, c=236.68 Å and β=96.3° and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4₂2₁2 with cell dimensions a=b=170.2 Å, c=156.9 Å and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 Å respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.     

Low resolution crystal structure of hagfish insulin

Insulin from the Atlantic hagfish, Myxine glutinosa, crystallizes in space group P4₁2₁2 with a monomer in the asymmetric unit. The application of the Rossmann &; Blow (1962) rotation function, utilizing the known 2-zinc pig insulin crystal structure, has established the existence of an insulin dimer containing a crystallographic 2-fold axis. The position of the hagfish insulin molecule in the unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 Å resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.     

Crystallization and preliminary X-ray analysis of two different forms of mitochondrial creatine kinase from chicken cardiac muscle

Crystals of mitochondrial creatine kinase isolated from chicken heart were grown by precipitation with polyethylene glycol 1000. The enzyme has been crystallized in the absence and presence of ATP in two different space groups. Crystals are tetragonal, with space group P42(1)2, a = b = 171 A, c = 150 A in the absence of ATP; and P422, a = b = 101 A, c = 114.4 A in the presence of ATP. We suggest that there is one octamer (346 kDa) per asymmetric unit without ATP and one dimer (86 kDa) per asymmetric unit with ATP. Using synchrotron radiation, the octameric form diffracts to at least 3 A resolution.     

Crystallization and preliminary structural studies of a chorismate mutase catalytic antibody complexed with a transition state analog

The Fab′ fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 Å and are suitable for X-ray crystallographic studies. © 1994 John Wiley & Sons, Inc.     

Crystallization and preliminary X-ray analysis of the Escherichia coli replication terminator protein complexed with DNA

Crystals of the Escherichia coli replication terminator protein (Tus) complexed with its binding site DNA were obtained by a microdialysis method using PEG 4000. They belong to the tetragonal space group P4₁2₁2 or P4₃2₁2 with the unit cell parameter: a = 68.1 Å, c = 230.7 Å and contain one protein-DNA complex in an asymmetric unit. The native data set has been collected to 2.7 Å 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 ?, b= 117.0 ?, c = 184.0?, 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 ?. Form II crystal obtained by using PEG4000 as precipitant belongs to space group P6₃ with unit cell constants a = 155.0 ?, c = 40.3 ?, γ =120.0° and one(αβ) unit in the asymmetric unit. The crystals diffract beyond 2.9 ?. The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.     

Crystallization and preliminary X-ray diffraction studies of leishmanolysin,the major surface metalloproteinase from Leishmania major

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microhet-erogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4₁2₁2 or the enantiomorph P4₃2₁2, has unit cell parameters of a = b = 63.6 Å, c = 251.4 Å, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 Å, b = 90.6 Å, c = 70.6 Å, β = 110.6°, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 Å resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress. © 1995 Wiley-Liss, Inc.