Crystallization of a dihydrolipoyl transacetylase--dihydrolipoyl dehydrogenase subcomplex and its implications regarding the subunit structure of the pyruvate dehydrogenase complex from Escherichia coli.

A subcomplex consisting of dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase, two of the three enzymes comprising the Escherichia coli pyruvate dehydrogenase complex, has been crystallized. X-ray diffraction data establish that the space group is P2₁3 with unit cell dimension $\text{a=211 .5}\text{A}\text{?}$. The unit cell contains four molecules of the subcomplex, each possessing 3-fold crystallographic and molecular symmetry. This finding, together with biochemical and electron microscopic data reported elsewhere, establish unequivocally that dihydrolipoyl transacetylase, the core enzyme of the pyruvate dehydrogenase complex, consists of 24 identical subunits with octahedral (432) symmetry. In the case presented here, the 432 symmetry of the transacetylase is reduced to 3-fold symmetry in the subcomplex by the addition of dihydrolipoyl dehydrogenase subunits. Crystal density measurements indicate that the dihydrolipoyl transacetylase present in these crystals is considerably smaller than the core mass generally reported for intact transacetylase. The implications of these findings are discussed with respect to the subunit stoichiometry and structure of the E. coli pyruvate dehydrogenase complex.     

Structure of a bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola: crystallographic evidence for a trimer

Crystals of the bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola, previously described by J. M. Olson et al. (1969), have been re-examined by X-ray diffraction. The space group is P6₃, as reported in the earlier work, but revised cell dimensions, $\text{a = b = 112.4 ± 0.4}\text{A}\text{?}\text{, c = 98.4 ± 0.4}\text{A}\text{?}$, were obtained, leading to a unit cell volume one third of that reported previously. Correction of this error leads to the conclusion that the bacteriochlorophyll-protein complex must be a trimer consisting of three identical subunits arranged about a crystallographic symmetry axis. Also a new trigonal crystal form of the bacteriochlorophyll-protein has been obtained, and is consistent only with a molecule composed of three identical or near-identical subunits. Models of the molecular packing for both crystal forms are presented.The molecular weight of the bacteriochlorophyll-protein complex, determined from crystal density measurements, is (1.53 ± 0.23) × 10⁵, and the overall molecular dimensions are about 55 Å along the trimer axis, and 83 Å at right angles to this. There are probably seven bacteriochlorophyll molecules in each subunit.     

A tetragonal crystal form of horse spleen apoferritin and its relationship to the cubic modification

Horse spleen apoferritin has been crystallized as tetragonal plates and needles with a unit cell with $\text{a = b = 147 ± 0.5}\text{A}\text{?}\text{and}\text{c = 154.4 ± 0.5}\text{A}\text{?}$. The space group is P42₁2 and the unit cell contains two molecules in a pseudo-body-centred arrangement. The intensity distributions and calculated rotation functions of tetragonal and cubic crystals have been compared. The symmetry of the diffraction patterns from cubic crystals indicates that the molecules have 432 symmetry with their 4-fold axes lying along the cube axes. In the tetragonal crystals one molecular 4-fold axis lies parallel to c, the unique axis, while the rest of the molecular point symmetry is not used by the lattice. Instead the remaining 4-fold axes of the two molecules, which lie in planes perpendicular to c, are rotated ± 17.5 ° with respect to the tetragonal a axis. The finding that apoferritin reassembled from subunits can be crystallized in both tetragonal and cubic forms confirms its conformational similarity to native molecules.     

Crystals of a functional 70,000 molecular weight subunit of hemocyanin from Limulus polyphemus

Crystals have been obtained of a subunit of Limulus polyphemus hemocyanin. The blue crystals have the symmetry of the space group R32 with hexagonal lattice parameters $\text{a = 115}\text{a}\text{?}$, $\text{c = 285}\text{A}\text{?}$. There is one 70,000 molecular weight subunit per asymmetric unit. Each subunit contains two non-heme copper atoms and can reversibly bind one oxygen molecule.     

Crystallographic study of the anti-tumor protein ricin

The anti-tumor protein ricin (also called RCA_II) has been crystallized in a form suitable for X-ray diffraction analysis. The crystal symmetry is orthorhombic spacegroup P2₁2₁2₁ with $\text{a = 72.9, b = 79.1, c = 114.7}\text{A}\text{?}$ and a single ricin molecule of molecular weight 65,000 per asymmetric unit. The crystals diffract well and are stable in the X-ray beam. At least one useful heavymetal derivative has been found as part of a high-resolution study.     

Crystallization of tobacco ringspot virus

Tobacco ringspot virus belongs to the nepovirus group of the small spherical plant viruses. It has been suggested that the protein capsid, which displays icosahedral symmetry, consists of 240 protein subunits which are arranged on a T = 4 surface lattice. The virus crystallizes in orthorhombic space group P2₁2₁2₁ ($\text{a = 388}\text{A}\text{?}\text{, b = 396}\text{A}\text{?}\text{,}\text{and}\text{c = 405}\text{A}\text{?}$). The unit cell contains four virus particles while the crystallographic asymmetric unit consists of one complete virion. X-ray diffraction data from the crystals extend to at least 3.3 Å resolution.     

Crystallisation of tRNALeuCUG from Escherichia coli after purification with hydroxyapatite.

tRNA_CUG^Leu from E. Coli was purified by column chromatography on benzoylated DEAE-cellulose, followed by hydroxyapatite prepared by an improved method. Crystals obtained by vapour diffusion gave X-ray diffraction out to 7 Å in the hk0 projection and 10 Å in h0?. The space group was P4₂2₁2 with $\text{a = b = 133}\text{A}\text{?}$, $\text{c = 66}\text{A}\text{?}$ and 8 molecules in the unit cell. Birefringence showed preferred orientation of RNA helical regions in the ab plane.     

The structures of barium hexafluorosilicate and cesium hexafluororhenate(V)

The crystal structure of CsReF₆, together with a reinvestigation of that of BaSiF₆, is reported. Both have been determined from single crystal three-dimensional X-ray diffraction data. The structure of BaSiF₆ has been found to conform to the initially assigned space group R$\text{3}$m, contrary to the suggestions of other workers. The unit cell of BaSiF₆ has the dimensiona a_hex 7.189(1), c_hex 7.015(1) Å; Z = 3. Refinement by a least squares method gave R 0.0079 and R_w 0.0077. Crystals of CsReF₆ belong to the lower symmetry rhombohedral space group R$\text{3}$. The unit cell has the dimensions a_hex 7.853(1), c_hex 8.140(1) Å; Z = 3. Refinement gave R 0.031 and R_w 0.030. The lowering of symmetry is caused by rotation of the ReF₆^? octahedra about the 3-fold axis through each Re atom, causing CsReF₆ to have the KOsF₆ structure.     

Crystallographic study on the orthorhombic crystal of l-asparaginase from Escherichia coli HAP

The orthorhombic crystals of l-asparaginase from Escherichia coli A-1-3 KY3598 were characterized by the X-ray diffraction method as belonging to the space group P2₁22₁ with unit cell dimensions $\text{a = 116.7}\text{A}\text{?}\text{, b = 62.9}\text{A}\text{?}\text{and}\text{c = 86.6}\text{A}\text{?}$. The crystals contain one half of the tetrameric enzyme molecule per asymmetric unit, the smallest so far reported for this enzyme. Preliminary analysis of the Patterson map indicated that the molecule had at least pseudo 222 symmetry and established the position of the centers of the molecules in the unit cell.     

Preliminary X-ray data for the ribose binding protein from Salmonella typhimurium

Crystals of the periplasmic ribose binding protein of Salmonella typhimurium have been subjected to X-ray analysis. The crystals grow as rectangular parallelopipeds with the symmetry of space group P2₁. Unit cell dimensions are $\text{a = 64·4}\text{A}\text{?}\text{, b = 60·6}\text{A}\text{?}\text{, c = 62·8}\text{A}\text{?}$, and β = 91·25 °. There are two molecules of molecular weight 29,000 per asymmetric unit.     

Letter: Crystallization and preliminary x-ray diffraction studies on tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus

Conditions have been established for the crystallization of tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus at room temperature. The crystals are extremely well-ordered, exhibiting diffraction spots out to at least 2.7 Å, and can be grown to a convenient size for X-ray crystallographic analysis. The crystals are trigonal with a space group P3₁21, the unit cell having dimensions of $\text{a = 64.4}\text{A}\text{?}$ and $\text{c = 238}\text{A}\text{?}$; the crystallographic asymmetric unit is probably one subunit of the dimeric (2 × 45,000, mol. wt) enzyme. The enzyme crystals are extremely stable and exhibit good resistance to radiation damage. This amino-acyl-tRNA synthetase appears to be amenable to complete structure determination by X-ray crystallography.     

Highly ordered crystals of the plant seed protein crambin.

Crystals of crambin, a plant seed protein of molecular weight 5000, diffract X-rays strongly to the interplanar spacing limit of 0.88 Å. These diffraction data should allow a definition of atomic structure that is on a par with that typically obtained from crystals of small organic molecules. The crystals are in space group P2₁ and have unit cell dimensions $\text{a = 41.1}\text{A}\text{?}\text{, b = 18.7}\text{A}\text{?}\text{, c = 22·7}\text{A}\text{?}\text{,}\text{and}\text{β = 90.6 °}$. The asymmetric unit contains one protein molecule.     

Electron diffraction determination of chain packing of a ketonic insect wax

Single crystal electron diffraction patterns are obtained from thin microcrystals of two polymorphic forms of a pure ketonic wax secreted by the wooly alder aphid, Prociphilus tessellatus. One crystalline form gives intensity data which agree well with the commonly observed O_⊥ methylene subcell. The unit cell constants for the projection are $\text{a = 7.52}\text{A}\text{?}$ and $\text{b = 5.05}\text{A}\text{?}$. The other polymorph, which is found less often, is a rectangular supercell with pseudohexagonal symmetry in the intensity-weighted hk0 reciprocal net. The supercell parameters in projection are $\text{a = 42.06}\text{A}\text{?}$, and $\text{b = 9.12}\text{A}\text{?}$ with the inner order hexad of intense spots occurring at 4.20 Å.     

The crystal structure of uncomplexed-hydrated cyclooctaamylose

Cyclooctaamylose crystallizes from aqueous solution with space-group symmetry P2₁ and lattice parameters: $\text{a}\text{= 20.253(8),}\text{b}\text{= 10.494(5),}\text{c}\text{= 16.892(6)}$ A and β = 105.32(1)^o, Z = 2; the apparent formular per asymmetric unit is C₄₈H₈₀O₄₀·17H₂O. The macrocycle is in an open conformation but displays significant deviations from ideal eight fold molecular symmetry. Of the 19 water molecules thus far located, four of which have occupancy factors of one half, 12 may be characterized as being in the torus of the cycloamylose.     

Purification and structural properties of the precursors of the globin messenger RNAs

X-ray diffraction data have been obtained from sodium and calcium salts of a proteoglycan rich in chondroitin 4-sulfate isolated from the Swarm rat chondrosarcoma. When sodium is the only countercation associated with the proteoglycan, the oriented polysaccharide chains adopt a 3-fold helical conformation in the solid state and pack in a trigonal unit cell with dimensions a = b = 1.45 nm and c = 2.88 nm. Addition of small amounts of calcium or full conversion of the polyanion from a sodium to a calcium salt form results in a conformational transition to a somewhat more extended 2-fold structure.For the calcium salt X-ray intensity data were used to refine the polysaccharide conformation and packing arrangement in the unit cell. Two antiparallel chains were found to crystallize in an orthorhombic unit cell with space group P22₁2₁ and dimensions a = 0.745 nm, b = 1.781 nm and c = 1.964 nm. The individual helix axes intersect the base plane of the unit cell at (x_f = 0, y_f = 0) and ($\text{x}{}_{\mathrm{f}}\text{= 0, y}{}_{\mathrm{f}}\text{=}\text{1}\text{2}$), and the polyanions are crystallographically equivalent, being related by the symmetry of the space group.The conformation of chondroitin 4-sulfate is stabilized intramolecularly by O.3 … O.5 hydrogen bonds across the β(1 → 4) linkage as well as by OSO^?₃ … Ca²⁺ … ^?OOC co-ordination across the β(1 → 3) linkage. Within the lattice adjacent parallel chains interact through COO^? … Ca²⁺ … ^?OOC bridges, and each calcium co-ordination shell is completed with an additional five water molecules to form a distorted, square antiprism. These water molecules are hydrogen-bonded to neighboring polyanions, and all intermolecular interactions involve water bridges or calcium ion co-ordination.On the basis of the refined packing model and the known structural features of the proteoglycan, models are considered for proteoglycan organization in connective tissue. Consideration of the conformational directing influence and relative abundance of calcium in the intercellular matrix suggest that the secondary structure of chondroitin 4-sulfate in vivo is likely to be similar to the conformation described in this study.     

Crystal data, molecular dimensions and molecular symmetry in cytochrome oxidase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa cytochrome oxidase (nitrite reductase, cytochrome cd) has been crystallized in space group P2₁2₁2 with cell dimensions $\text{a = 122.8}\text{A}\text{?}\text{, b = 87.2}\text{A}\text{?}\text{, c = 73.4}\text{A}\text{?}$. Density measurements suggest that the asymmetric unit contains one 63,000 molecular weight subunit of the dimeric molecule. Crystal data agree well with electron microscopy of single molecules. The X-ray pattern extends beyond 2.5 Å resolution, and structure analysis is in progress.     

Identification of the regulatory subunit of a cAMP-dependent protein kinase in Dictyostelium discoideum

The occurrence of a cytosolic cAMP-binding protein of an approximate molecular weight of 41,000 daltons was monitored in vegetative and developing amoebae of $\text{Dictyostelium}\text{discoideum}$ by the use of the photoaffinity probe (³²P) 8N₃-cAMP. There was a large apparent increase in the amount of this binding protein during development; its molecular weight remained constant, if appropriate methods were employed for the disruption of the amoebae. Comigration during electrophoresis on two-dimensional gels identifies this cAMP-binding protein, photoaffinity-labeled in crude extracts, as the regulatory subunit of the cAMP-dependent protein kinase of $\text{D.}\text{discoideum}$.     

Endogenous, cyclic 3'-5' AMP-dependent phosphorylation of human red cell pyruvate kinase.

Ribosomal proteins of $\text{Physarum}\text{polycephalum}$ were labelled $\text{in}\text{vivo}$ with ³²PO₄. Three acid phosphoproteins were observed in the large subunit, while two basic ones were present in the small subunit. Ribosomal phosphoprotein S3 accounted for 70% of the total radioactivity and may be equivalent to S6 from rat liver.     

A preliminary crystallographic investigation of rabbit muscle creatine kinase

Three crystal forms of rabbit muscle creatine kinase have been grown, one of which seems suited to a high resolution X-ray diffraction study. The first form is of monoclinic space group P2₁ with $\text{a = 54}\text{A}\text{?}\text{, b = 114}\text{A}\text{?}\text{, c = 145}\text{A}\text{?}\text{, β = 91 °}$ and has as the asymmetric unit two molecules of total molecular weight 160, 000. The second form, grown in the presence of mercurials, is of space group A2 with $\text{a = 52}\text{A}\text{?}\text{, b = 165}\text{A}\text{?}\text{, c = 237}\text{A}\text{?}\text{, β = 91 °}$ and also has two molecules in the asymmetric unit. The third crystal form, grown in the presence of a high concentration of cysteine, is of apparent space group P2₁2₁2₁, but evidence indicates that the true space group may be P2₁22₁. The dimensions of the orthorhombic unit cell are $\text{a = 47}\text{A}\text{?}\text{, b = 86}\text{A}\text{?}\text{, c = 125}\text{A}\text{?}$, and the asymmetric unit contains a single protein subunit. Assuming the latter space group, then the creatine kinase molecule possesses a twofold axis relating two identical subunits.     

Purification and preliminary crystallographic studies on azurin and cytochrome c' from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015.

Purification and crystallisation procedures are reported for azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015. The azurin crystals from A. denitrificans are suitable for high-resolution X-ray structure analysis. They are orthorhombic, space group C222₁ (with marked tetragonal pseudo-symmetry), cell dimensions $\text{a = 75.0}\text{A}\text{?}\text{, b = 74.1}\text{A}\text{?}\text{, c = 99.5}\text{A}\text{?}$, with two molecules per asymmetric unit. The cytochrome c′ crystals from both species are hexagonal, space group P6₁22 (or P6₅22), cell dimensions $\text{a = b = 54.7}\text{A}\text{?}\text{, c ~ 185}\text{A}\text{?}$, γ = 120 °, with one subunit (molecular weight 14,000) in the asymmetric unit.