Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from <Emphasis Type="Italic">Eisenia fetida</Emphasis>

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida, a protein functioning not only as a direct fibrinolytic enzyme, but also as a plasminogen activator, has been crystallized in P212121 space group with 3 proteinmolecules per asymmetric unit. Four heavy atom derivatives were prepared using a mother liquor containing 1.4 mol@L-1 Li2SO4 and 0.1 mol@L-1 MOPS buffer (pH7.2) and used to solve the protein's diffraction phase. The heavy atom binding sites in the derivative crystals were determined using difference Patterson and difference Fourier methods and were refined in combination to yield the initial protein's structure phase at 0.25 nm resolution. The non-crystallographic symmetryrelationship of the three independent protein molecules in the asymmetric unit was determined using the correlative heavy atom sites and used for the averagingof the initial electron density. As a result, the electron density was significantly improved, providing a solid foundation for subsequent structure determination.     

Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida, a protein functioning not only as a direct fibrinolytic enzyme, but also as a plasminogen activator, has been crystallized in P2₁2₁2₁ space group with 3 protein molecules per asymmetric unit. Four heavy atom derivatives were prepared using a mother liquor containing 1.4 mol · L^-1 Li₂SO₄ and 0.1 mol · L^-1 MOPS buffer (pH7.2) and used to solve the protein’s diffraction phase. The heavy atom binding sites in the derivative crystals were determined using difference Patterson and difference Fourier methods and were refined in combination to yield the initial protein’s structure phase at 0.25 nm resolution. The non-crystallographic symmetry relationship of the three independent protein molecules in the asymmetric unit was determined using the correlative heavy atom sites and used for the averaging of the initial electron density. As a result, the electron density was significantly improved, providing a solid foundation for subsequent structure determination.     

Crystallization and preliminary X-ray characterization of a soybean seed lipoxygenase

An isoenzyme of soybean (Glycine max L. Merrill cv. Provar) lipoxygenase (EC 1.13.11.12) has been crystallized using the vapor diffusion method. Crystals were grown from solutions of the protein (7 mg/ml) using 10 to 20% (w/v) polyethylene glycol 8000 in citrate/phosphate buffer (pH 5.7) containing 0.5% (w/v) n-octyl-beta-D-glucopyranoside. The crystals reached maximum dimensions of 0.3 mm x 0.2 mm x greater than 2 mm. The enzyme crystallized in space group C222(1) with unit cell dimensions a = 246 A, b = 193 A and c = 75 A. A calculated Vm value of 2.35 A3/dalton was obtained assuming two molecules per asymmetric unit. The density of the crystals was found to be 1.16 g/ml, which confirmed the presence of two molecules per asymmetric unit and indicated a solvent content of 47.5%.     

The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5'-phosphate-dependent enzyme.

The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P2(1)2(1)2. The unit cell has dimensions a = 76.0 A, b = 138.3 A, c = 93.5 A and it contains two subunits of the tetrameric molecule in the asymmetric unit. Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanner. The structure has been solved at 2.7 A resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non-crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases.     

Twinning in crystals of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase

The coenzyme-bound form of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase has been shown to crystallize in the space group C2 and not C222₁ as previously reported. The unit cell contains two tetrameric molecules with the dimer of molecular weight 72,000 as the crystallographic asymmetric unit. The recorded X-ray intensity distribution clearly indicates the presence of non-crystallographic 2-fold axes perpendicular to the crystallographic 2-fold axis showing that the subunits are arranged with near perfect 222 symmetry.Isomorphous derivatives of the enzyme have been prepared and the heavy atom positions defined in complete agreement with the C2 space group assignment. Further confirmation that the space group is C2 and not C222₁ comes from the 3.5 Å resolution electron density map of the human enzyme, which appears almost identical to that of the lobster holo-enzyme where no such space group ambiguity exists.     

江浙蝮蛇毒溶血毒素晶体培养及初步晶体学研究

从江浙蝮蛇中分离纯化的碱性磷脂酶Ａ２在ｐＨ９．５,０．０５ｍｍｏｌ／ＬＣＨＥＳ缓冲液中,用汽相悬滴扩散的方法,获得了适用于高分辨率Ｘ射线结构分析的单晶．经Ｘ２００Ｂ面探测器分析,表明该晶体属于正交晶系,Ｐ２Ｉ２Ｉ２Ｉ空间群,晶胞参数为ａ＝９７．１３,ｂ＝１０３．６９,ｃ＝２３．２７．并收集了一套衍射数据,独立衍射点数１２００１个,数据完整度为８６．２％,Ｒｍｅｒｇｅ为０．０４５９．最高分辨率达２．０,根据分子量与晶胞体积估算,一个不对称单位含两个分子．     

Crystal structure of a novel non-Pfam protein PF2046 solved using low resolution B-factor sharpening and multi-crystal averaging methods

Sometimes crystals cannot diffract X-rays beyond 3.0 ? resolution due to the intrinsic flexibility associated with the protein. Low resolution diffraction data not only pose a challenge to structure determination, but also hamper interpretation of mechanistic details. Crystals of a 25.6 kDa non-Pfam, hypothetical protein, PF2046, diffracted X-rays to 3.38 ? resolution. A combination of Se-Met derived heavy atom positions with multiple cycles of B-factor sharpening, multi-crystal averaging, restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5 (R_free=24.7). The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers. Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.     

Structure of yeast hexokinase. II. A 6 angstrom resolution electron density map showing molecular shape and heterologous interaction of subunits

An electron density map of yeast hexokinase has been calculated at 6 Å resolution using six heavy atom derivatives. The map shows each of the enzyme's two 51,000 molecular weight subunits to consist of two separate lobes connected by a narrow bridge of density. Furthermore, these two subunits are related to each other in the asymmetric unit of the crystal by a quasi-2-fold rather than a true 2-fold axis. That is, they are related by a rotation of 180 ° plus a relative translation of 3.6 Å along the symmetry axis. This gives rise to a heterologous subunit interaction and a possibility of non-identical structure and function for these chemically identical subunits. The molecule is quite asymmetric, having dimensions of 150 Å × 45 Å × 55 Å. Each subunit is about 80 Å × 40 Å × 50 Å.A portion of an electron density map at 3 Å resolution has been also calculated, based on phases from two heavy atom derivatives. Polypeptide backbone and side chains are visible in this map.     

Preliminary crystallographic data and quaternary structural implications of the central subunit of the multi-subunit complex transcarboxylase

The hexameric central subunit (Mr = 360,000) of the multi-subunit complex transcarboxylase has been crystallized by bulk dialysis against 250 mM-sodium acetate (pH 5.5). The crystals are cubic, a = 193.1 A, space group P4(1)32 or enantiomorph. The number of molecules per unit cell is four and was deduced from the density of the crystals (1.10 g cm-3) and the mother liquor (1.01 g cm-3) and the specific volume of the protein calculated from molecular dimensions obtained from electron microscopy studies. Four molecules per cell requires the central subunits to lie on 3-fold axes, which are perpendicular to 2-fold rotation axes, so that the molecules satisfy 32 symmetry giving one subunit as the asymmetric unit. Of the four possible models that have been considered for the quaternary structure of transcarboxylase, only that with antiparallel subunits, two sets of isologous binding sites and D3 symmetry is in agreement with the symmetry requirements of the cubic crystals.     

The structure of 2Zn pig insulin crystals at 1.5 A resolution

The paper describes the arrangement of the atoms within rhombohedral crystals of 2Zn pig insulin as seen in electron density maps calculated from X-ray data extending to 1.5 A (1 A = 10(-10) m = 10(-1) nm) at room temperature and refined to R = 0.153. The unit cell contains 2 zinc ions, 6 insulin molecules and about 3 x 283 water molecules. The atoms in the protein molecules appear well defined, 7 of the 102 side chains in the asymmetric unit have been assigned alternative disordered positions. The electron density over the water molecules has been interpreted in terms of 349 sites, 217 weighted 1.0, 126 weighted 0.5, 5 at 0.33 and 1 at 0.25 giving ca. 282 molecules. The positions and contacts of all the residues belonging to the two A and B chains of the asymmetric unit are shown first and then details of their arrangement in the two insulin molecules, 1 and 2, which are different. The formation from these molecules of a compact dimer and the further aggregation of three dimers to form a hexamer around two zinc ions, follows. It appears that in the packing of the hexamers in the crystal there are conflicting influences; too-close contacts between histidine B5 residues in neighbouring hexamers are probably responsible for movements of atoms at the beginning of the A chain of one of the two molecules of the dimer that initiate movements in other parts, particularly near the end of the B chain. At every stage of the building of the protein structure, residues to chains of definite conformation, molecules, dimers, hexamers and crystals, we can trace the effect of the packing of like groups to like, aliphatic groups together, aromatic groups together, hydrogen-bonded structures, positive and negative ions. Between the protein molecules, the water is distributed in cavities and channels that are continuous throughout the crystals. More than half the water molecules appear directly hydrogen bonded to protein atoms. These are generally in contact with other water molecules in chains and rings of increasing disorder, corresponding with their movement through the crystals. Within the established crystal structure we survey next the distribution of hydrogen bonds within the protein molecules and between water and protein and water and water; all but eight of the active atoms in the protein form at least one hydrogen bond.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution

The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods. The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177. The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules. The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry. The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice. A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map. Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis. This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources. In the crystal both of the anion binding sites are occupied by sulphate ions. The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule.     

Low resolution crystal structures of Taka-amylase A and its complexes with inhibitors.

The molecular structure of Taka-amylase A, an alpha-amylase from Aspergillus oryzae, has been studied at 6 A resolution by X-ray diffraction analysis. The electron density map showed a non-crystallographic three-fold screw arrangement of the molecules in the crystal. The molecule is an ellipsoid with approximate dimensions of 80 x 45 x 35 A and contains a hollow which may correspond to the active center. The inhibitor molecules bind to Taka-amylase A at four different sites, one of which is located in the hollow of the enzyme. The probable position of a thiol group is discussed in connection with heavy atom binding.     

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2.5 A resolution.

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.     

Three-dimensional structure of Gln25-ribonuclease T1 at 1.84-A resolution: structural variations at the base recognition and catalytic sites.

The structure of the Gln25 variant of ribonuclease T1 (RNase T1) crystallized at pH 7 and at high ionic strength has been solved by molecular replacement using the coordinates of the Lys25-RNase T1/2'-guanylic acid (2'GMP) complex at pH 5 [Arni et al. (1988) J. Biol. Chem. 263, 15358-15368] and refined by energy minimization and stereochemically restrained least-squares minimization to a crystallographic R-factor of 14.4% at 1.84-A resolution. The asymmetric unit contains three molecules, and the final model consists of 2302 protein atoms, 3 sulfates (at the catalytic sites), and 179 solvent water molecules. The estimated root mean square (rms) error in the coordinates is 0.15 A, and the rms deviation from ideality is 0.018 A for bond lengths and 1.8 degrees for bond angles. Significant differences are observed between the three molecules in the asymmetric unit at the base recognition and catalytic sites.     

Structural analysis of T4 DNA helix destabilizing protein (gp32 I) crystal by electron microscopy

Low dose electron diffraction and imaging techniques have been applied to the study of the crystalline structure of gp32*I, a DNA helix destabilizing protein derived from bacteriophage T4 gene 32 protein. A quantitative analysis of intensities from electron diffraction patterns from tilted, multilayered gp32*I crystal has provided the unit cell thickness of the crystal. The three-dimensional phases indicate that the space group P2(1)2(1)2. By taking into account the unit cell volume and the solvent content in the crystal, it was deduced that there is one gp32*I molecule in each asymmetric unit. A projected density map of unstained, glucose-embedded gp32*I crystal was synthesized with amplitudes from electron diffraction intensities and phases from electron images with reflections out to 7.6 A. Because of the similarity in the scattering density between glucose and protein, this projected map cannot be interpreted with certainty. A low resolution three-dimensional reconstruction shows that the protein molecule is about 90 A long and about 20 A in diameter. Because the dimer is formed around a dyad axis, the protein molecules comprising it must be arranged head-to-head. This dimeric arrangement of the proteins in the unit cell may be implicated as one of the conformational states of this protein in solution.     

Crystallographic studies of the chicken gizzard G-actin X DNase I complex at 5A resolution

The structure of the chicken gizzard G-actin X DNase I complex has been determined at 5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using four heavy atom derivatives. The mean figure of merit was 0.65. Dimensions of the three molecular species, the complex, G-actin and DNase I, were determined based on the "cypress wood" models derived from the electron density map. The natures of the heavy atom binding sites are discussed in relation to the distinction between the two component molecules. The pattern of successive contacts between actin molecules observed in the present crystal seems unrelated to that found in F-actin.     

The three-dimensional structures of methanol dehydrogenase from two methylotrophic bacteria at 2.6-A resolution.

The structures of methanol dehydrogenase (MEDH) from two closely related methylotrophic bacteria, Methylophilus methylotrophus and W3A1, have been determined at 2.6-A resolution. The molecule, a quinoprotein of molecular mass of about 138 kDa, contains two heavy (H) and two light (L) subunits of unknown sequence and two molecules of noncovalently associated pyrroloquinoline quinone. The two enzymes crystallize isomorphously in space group P2(1) with one H2L2 heterotetramer in the asymmetric unit. The electron density map of the M. methylophilus enzyme was obtained by multiple isomorphous replacement with anomalous scattering and improved by solvent leveling and electron density averaging. For model building, the amino acid sequence of MEDH from Paracoccus denitrificans for the H subunit and from Methylobacterium extorquens AM1 for the L subunit were used to represent the unknown amino acid sequence. At the present time, 579 and 57 amino acid residues for the large and small subunits, respectively, have been fitted into the map. The phases for MEDH from M. methylophilus were used directly to analyze the W3A1 structure, and both structures were refined to R-factors (where R = sigma[Fo-Fc[/sigma Fo) of 0.277 and 0.266, respectively. The L subunit contains a long alpha-helix and an extended N-terminal segment, both lying on the molecular surface of the H subunit. The H subunit contains eight antiparallel beta-sheets, each consisting of four strands arranged topologically like the letter W. The eight Ws are arranged circularly, forming the main disc-shaped body of the subunit, with some short helices and loops connecting the consecutive Ws, as well as some excursions within and between some of the Ws. The pyrroloquinoline quinone prosthetic group is located in the central channel of the large subunit near the surface of the molecule. The topology of the eight-W folding unit is similar to those of the six- and seven-W folding units previously reported for three other proteins, neuraminidase, methylamine dehydrogenase, and galactose oxidase.     

The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes

The crystal structure of xylose isomerase [E.C. 5.3.1.5] from Streptomyces olivochromogenes has been determined to 3.0 A resolution. The crystals belong to space group P22(1)2(1) with unit cell parameters a = 98.7, b = 93.9, c = 87.7. The asymmetric unit contains half of a tetrameric molecule of 222 symmetry. The two-fold axis relating the two molecules in the asymmetric unit is close to where a crystallographic two-fold would be if the space group were I222. This causes the diffraction pattern to have strong I222 pseudo-symmetry, so all data were collected in this pseudo-space group. Since the sequence of this enzyme has not been reported, a polyalanine backbone has been fitted to the electron density. Xylose isomerase has two domains: the N-terminal domain is an eight-stranded alpha/beta barrel of 299 residues. The C-terminal domain is a large loop of 50 residues which is involved in intermolecular contacts. Comparison of xylose isomerase with the archetypical alpha/beta barrel protein, triose phosphate isomerase, reveals that the proteins overlap best when the third (alpha beta) strand of xylose isomerase is superimposed on the first (alpha beta) strand of triose phosphate isomerase. This same overlap has also been found between the muconate lactonising enzyme and triose phosphate isomerase [Goldman et al. (1987) J. Mol. Biol., in press].     

A crystallographic model for azurin a 3 A resolution.

The structure of the blue copper protein azurin (M_r 14,000) from Pseudomonas aeruginosa has been determined from a 3.0 Å resolution electron density map computed with phases based on a uranyl derivative to 3 Å resolution and a platinum derivative to 3.7 Å. Interpretation of the somewhat noisy map was based on comparison of the density of the four molecules in the asymmetric unit with their averaged density. The polypeptide chain folds into an eight-strand β barrel with an additional flap containing a short helix. The copper atom is bound at one end and on the inside of the barrel, probably to a cysteine, a methionine, and two histidine residues.     

Crystals of native and modified bovine rhodopsins and their heavy atom derivatives

Rhodopsin, the pigment protein responsible for dim-light vision, is a G protein-coupled receptor that converts light absorption into the activation of a G protein, transducin, to initiate the visual response. We have crystallised detergent-solubilised bovine rhodopsin in the native form and after chemical modifications as needles 10-40 microm in cross-section. The crystals belong to the trigonal space group P3(1), with two molecules of rhodopsin per asymmetric unit, related by a non-crystallographic 2-fold axis parallel with the crystallographic screw axis along c (needle axis). The unit cell dimensions are a=103.8 A, c=76.6 A for native rhodopsin, but vary over a wide range after heavy atom derivatisation, with a between 101.5 A and 113.9 A, and c between 76.6 A and 79.2 A. Rhodopsin molecules are packed with the bundle of transmembrane helices tilted from the c-axis by about 100 degrees . The two molecules in the asymmetric unit form contacts along the entire length of their transmembrane helices 5 in an antiparallel orientation, and they are stacked along the needle axis according to the 3-fold screw symmetry. Hence hydrophobic contacts are prominent at protein interfaces both along and normal to the needle axis. The best crystals of native rhodopsin in this crystal form diffracted X-rays from a microfocused synchrotron source to 2.55 A maximum resolution. We describe steps taken to extend the diffraction limit from about 10 A to 2.6 A.