Single crystals of dextran: High temperature polymorph

Lamellar single crystals of a high temperature polymorph of synthetic dextran were prepared at temperatures ranging from 120 to 200°C in a mixture of water and polyethylene glycol. Individual crystals with lath-like shapes gave well resolved electron diffraction diagrams from which the reciprocal unit cell parameters a^*, b^* and γ^* could be measured. The direct cell parameters were then determined from a series of electron diffraction diagrams obtained by sequential tilting of the crystal about the b^* axis. This gave a = 0·922 ± 0·001 nm, b = 0·922 ± 0·001 nm, c (chain axis) = 0·78 ± 0·01 nm, α = γ = 90° and β = 91·3° ± 0·5°. The crystal symmetry was P2₁ with b as the unique monoclinic axis. These data coupled with the observed density of the crystals, indicated that the unit cell contained two antiparallel dextran chains of two residues each. When the crystals were grown at temperatures between 90 and 120°C, a percentage of crystals containing both low and high temperature polymorphs were obtained. These mixed crystals had most likely grown in syntaxy.     

Structural studies on triacetates of mannan and glucomannan

Mannan triacetates prepared from material extracted from ivory nut and Tubera salep were studied by means of electron and X-ray diffraction. The former is uniquely constituted of acetylated d-mannopyranosyl units linked by a (1 → 4)-β-linkage whereas the latter contains acetylated (1 → 4)-β-d-glucopyranosyl randomly distributed in the backbone with a ratio of mannose to glucose of about 3:1. However, there seems to be no effect on crystallisation due to the presence of the glucosidic units on the conformation of the chain.Single crystals of ivory nut triacetate were prepared by slowly cooling a dilute solution of nitromethane and butanol. The crystals were long narrow laths which provide electron diffraction data after annealing at 190°C in a vacuum.Two different unit cells were derived from the acetylated Tubera salep X-ray data. A first unit cell with a = 1·18 nm, b = 1·54 nm and c = 1·60 nm contains eight sugar units, whereas the second unit cell with a = 0.369 nm, b = 0·96 nm and c = 1·58 nm would accommodate 16 residues. The latter agrees best with the base-plane parameters derived from electron diffraction of single crystals.The X-ray fibre diagram was interpreted in terms of a two-fold helix and an asymmetric unit composed of two triacetyl mannopyranosyl units. This means that two chemically identical mannose units would not be conformationally equivalent along the backbone.The presence of glucose units in the backbone does not seem to perturb the crystalline conformation. The ‘isomorphous replacement’ hypothesis was invoked to explain this observation. The helical parameters derived herein for Tubera salep mannan triacetate are different from those reported earlier for the same acetylated glucomannan but crystallised using a different technique. This is attributed to the occurrence of polymorphism in this material.     

Electron microscopic studies on microcrystals of D-ribulose-1,5-biphosphate carboxylase from Dasycladus clavaeformis roth (Ag.)

Crystals of D-ribulose-1,5-biphosphate carboxylase (E.C. 4.1.1.39), naturally occurring in the extraplasmatic space of the unicellular green algae $\text{Dasycladus clavaeformis}$ (Dasycladaceae), were studied by means of electron microscopy and optical diffraction. Optical diffraction patterns were obtained from thin sections. It is shown that the crystals are composed of cubic unit cells with α ～ 31.5 nm. The density of the crystals was estimated as 1.07 ± 0.005 g/ml, a value that gives evidence of the presence of 12 enzyme molecules per unit cell. Optical diffraction studies of the thin sectioned crystals revealed 4mm -symmetry with four 2-fold rotation axes, resulting in at least a 222-symmetry.     

Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga

Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P222₁ with unit cell constants of a = 146.8 Å, b = 176.9 Å, and c = 353.7 Å, and the other one had a space group of P2₁2₁2₁ with unit cell constants of a = 209.2 Å, b = 237.5 Å, and c = 299.8 Å. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 Å resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 Å resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.     

Conformations and interactions of pectins: I. X-ray diffraction analyses of sodium pectate in neutral and acidified forms

X-ray diffraction patterns of uniaxially oriented, polycrystalline fibers of neutral sodium pectate can be indexed on the basis of an orthogonal unit cell with dimensions a = 0.84 nm, b = 1.43 nm, c (fiber axis) = 1.34 nm, which contains trisaccharide fragments of two polygalacturonic chains of opposite sense. The polysaccharide chains have 3₁ screw symmetry but are arranged in a lattice that has space group symmetry P2₁ (unique axis b). There are three sodium ions in each crystal asymmetric unit. They are all octahedrally co-ordinated to oxygen atoms of the galacturonan chains or of water molecules. Every oxygen atom is involved also in at least one hydrogen bond. Sodium pectate can be partially converted to pectic acid whose polysaccharide chains preserve the 3₁ pectate conformation, are packed in an orthogonal unit cell also with P2₁ symmetry but with quite different dimensions a = 0.99 nm, b (unique 2₁ axis) = 1.23 nm, c (fiber axis) = 1.33 nm. In this lattice, the polygalacturonic acid chains form corrugated sheets in which alternate molecules have opposite sense and are extensively hydrogen-bonded through their carboxyl groups.     

Letter to the editor: Electron diffraction for hydrated crystalline biopolymers: nigeran.

A method is presented to record electron diffraction diagrams on hydrated crystalline biopolymers. It involves quick-freezing of the hydrated crystals, which are maintained at a low temperature throughout the experiment. The technique is illustrated with lamellar single crystals of the linear polysaccharide nigeran. Water of hydration is shown to be in a sheet-like array and its removal is accompanied by development of lateral cracks. This morphological shrinkage coincides with an observed shortening of the b lattice parameter, which is normal to the lateral cracks.     

Bacteriophage P1 site-specific recombination. II. Recombination between loxP and the bacterial chromosome

Lipoprotein crystals of hagfish yolk platelets (Myxine glutinosa L.) are, by electron diffraction of embedded specimens, monoclinic (a > 19.8 nm, b > 8.9 nm, c > 9.0 nm, β ~ 105 °; space group C2: 2 dimers per cell). Using sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the four major protein bands in Myxine (molecular weights 30,000; 44,000: 80,000 and 130,000) correspond, with small differences, to similar bands of Xenopus yolk lipoproteins. The symmetric lipovitellin-phosvitin dimer in cyclostomes is unique and probably reflects the lack of diversification of homologous vertebrate protein species.     

Three-dimensional electron diffraction of plant light-harvesting complex

Electron diffraction patterns of two-dimensional crystals of light-harvesting chlorophyll a/b-protein complex (LHC-II) from photosynthetic membranes of pea chloroplasts, tilted at different angles up to 60°, were collected to 3.2 Å resolution at -125°C. The reflection intensities were merged into a three-dimensional data set. The Friedel R-factor and the merging R-factor were 21.8 and 27.6%, respectively. Specimen flatness and crystal size were critical for recording electron diffraction patterns from crystals at high tilts. The principal sources of experimental error were attributed to limitations of the number of unit cells contributing to an electron diffraction pattern, and to the critical electron dose. The distribution of strong diffraction spots indicated that the three-dimensional structure of LHC-II is less regular than that of other known membrane proteins and is not dominated by a particular feature of secondary structure.     

Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by x-ray diffraction and electron microscopy

Cytochrome oxidase from Pseudomonas aeruginosa has been crystallized from 2 m-ammonium sulfate. The crystals occur principally as thin diamond-shaped plates of space group P2₁2₁2 with unit cell dimensions of 92 Å × 115 Å × 76 Å. Determination of the density of glutaraldehyde-fixed, water-equilibrated crystals (1.167 g/cm³), coupled with the unit cell volume (804,000 ų), indicates that there is one subunit (~63,000 Mr) per asymmetric unit. X-ray diffraction data which were limited to 12 Å resolution due to small crystal size were obtained for the hk0 and 0kl zones using precession photography. Amplitude and phase data for the hk0, 0kl, and h0l zones were obtained from computer-based Fourier analysis of appropriate micrographs recorded from negatively stained microplates and thin sections of larger crystals using minimal beam electron microscopy. For crystals embedded in the presence of tannic acid it was possible to achieve 20 Å resolution which is comparable to the resolution achieved with negative staining of thin crystalline arrays. In addition, unstained electron diffraction on glutaraldehyde-fixed, glucose-stabilized plates was recorded to a resolution of 9 Å. The three-dimensional packing of the cytochrome oxidase dimer in the unit cell has been deduced from computer reconstructed images of the three principal projections along the crystallographic axes. The cytochrome oxidase dimer is located in the unit cell with the dimer axis coincident with a crystallographic 2-fold axis; thus within the resolution of the present data in projection (9 Å) the two subunits are identical, in agreement with biochemical evidence. The crystals have been prepared with the enzyme in the fully oxidized state and upon reduction a progressive cracking of the crystals is observed, possibly due to a conformational change dependent on the oxidation state of the heme iron.     

Organization of nucleosomes and spacer DNA in chromatin fibres

In this paper we analyse in detail the orientation of X-ray diffraction diagrams obtained from the following materials: nucleosome cores, whole nuclei and the sodium and thallium salts of H1-depleted nucleohistone and of briefly digested chromatin. Our analysis indicates that spacer DNA is organized in bundles of parallel segments which contribute to the equatorial maxima in the diagrams. Several models are compatible with this organization, in particular a modified solenoid model in which the central part is filled with such a bundle of spacer DNA segments parallel to the axis of the fibre. It is also shown that spacer DNA is covered by histones, probably the N-terminal regions. This observation indicates that the differential activity of nucleases on chromatin is strongly influenced by conformational features of DNA. An analysis of the orientation of the low angle rings found in the X-ray diffraction patterns of H1-depleted nucleohistone shows that the 11 nm peak has maxima which are ～ 0.007 nm^?1 off the meridian. The 5.5 and 8 nm peaks have a meridional maximum plus two side maxima which occur at spacings between 0.02 and 0.055 nm^?1 from the meridian, depending on the conditions. A comparison of these results with those reported by Finch et al.¹ for crystals indicates that in fibres the nucleosome cores are arranged with their short axis perpendicular to the axis of the fibre. Some evidence on the path of DNA in the nucleosome cores is also obtained.     

The molecular and crystal structure of dextrans: a combined electron and X-ray diffraction study. II. A low temperature, hydrated polymorph

The crystal and molecular structure of a dextran hydrate has been determined through combined electron and X-ray diffraction analysis, aided by stereochemical model refinement. A total of 65 hk0 electron diffraction intensities were measured on frozen single crystals held at the temperature of liquid nitrogen, to a resolution limit of 1.6 A. The X-ray intensities were measured from powder patterns recorded from collections of the single crystals. The structure crystallizes in a monoclinic unit cell with parameters a = 25.71 A, b = 10.21 A, c (chain axis) = 7.76 A and beta = 91.3 degrees. The space group is P2(1) with b axis unique. The unit cell contains six chains and eight water molecules, with three chains of the same polarity and four water molecules constituting the asymmetric unit. Along the chain direction the asymmetric unit is a dimer residue; however, the individual glucopyranose residues are very nearly related by a molecular 2-fold screw axis. The conformation of the chain is very similar to that in the anhydrous structure, but the chain packing differs in the two structures in that the rotational positions of the chains about the helix axes (the chain setting angles) are considerably different. The chains still pack in the form of sheets that are separated by water molecules. The difference in the chain setting angles between the anhydrous and hydrate structures corresponds to the angle between like unit cell axes observed in the diffraction diagrams recorded from hybrid crystals containing both polymorphs. Despite some beam damage effects, the structure was determined to a satisfactory degree of agreement, with the residuals R'(electron diffraction) = 0.258 and R(X-ray) = 0.127.     

Crystal structure of polyglycine I

An electron diffraction study has been made of oriented polyglycine I (the β modification of polyglycine) and of single crystals grown from solution. The unit cell is very similar to that postulated by Astbu?y (1949). It is monoclinic with parameters a = 9.54 Å, b(chainaxis) = 7.044 Å, c = 3.67 Å and β = 113°. Examination of the possible structures suggests that polyglycine I does not have the familiar antiparallel pleated sheet, but rather the closely related antiparallel rippled sheet structure first described by Pauling &; Corey (1953a).     

Purification,crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2₁, with unit cell dimensions a= 102.4 Å b= 116.5 Å c= 115.8 Å and β = 103.5 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 Å₃/Da. The crystals diffract to at least 2.3 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss, Inc.     

Hyaluronic acid: molecular conformations and interactions in two sodium salts.

A detailed structure for the tetragonal form (a = b = 0.989 nm, c, fibre axis, = 3.394 nm) of sodium hyaluronate has been obtained by analysing X-ray fibre diffraction data using new molecular modelling techniques. Two polysaccharide chains pass through each unit cell, one at the corner and one at the centre. The chains are anti-parallel to one another. Each chain is a left-handed, 4-fold helix of disaccharide units. There are intramolecular hydrogen bonds stabilising each glycosidic linkage. Octahedrally co-ordinated sodium ions link, by O … Na⁺ … O bridges, neighbouring polysaccharide chains that are further linked by hydrogen bonds. No double-helix model (as originally proposed for this structure) has been found to be free of unacceptable non-bonded contacts or to fit the diffraction intensities as closely.The tetragonal form, which is stable at zero relative humidity, contains no detectable water molecules. At higher relative humidities a related orthorhombic form is observed in which only the a dimension of the lattice is different (a = 1.153 nm, b = 0.989 nm, c = 3.386 nm). In this form the hyaluronate helix is 2-fold with tetrasaccharide units conformationally similar to the 4-fold helix of the tetragonal form. The Na⁺ … O binding and hydrogen bonds lost on expansion of the tetragonal lattice are all replaced in the orthorhombic structure by bridges through water molecules, four of which associated with each tetrasaccharide.     

Crystals of an antibody FV fragment against an integral membrane protein diffracting to 1.28 Å resolution

The F_v fragment of a monoclonal antibody, 7E2 (IgG₁, κ, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for highresolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 51.51 Å, b = 56.15 Å, c = 99.86 Å (1 Å = 0.1 nm) and contain one F _v fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 Å resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction study of 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea

1,3,8-Trihydroxynaphthalene reductase was crystallized in the presence of NADPH and the inhibitor tricyclazole. The crystals are trigonal, space group P3₁21 or its enantiomorph P3₂21. Two crystal forms with slightly different cell dimensions were obtained. Form A has unit cell dimensions a = b = 142.6 Å, c = 70.1 Å and form B cell dimensions a = b = 142.6 Å, c = 72.9 Å. The diffraction pattern of the latter crystal form extends to 2.5 Å resolution.     

Purification,crystallization, and preliminary X-ray studies of a bifunctional 5,10-methenyl/methylene tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenease), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a= 64.5 Å b= 84.9 Å c= 146.1 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 ų/Da. The crystals diffract to at least 2.8 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 Å resolution native data set has been collected at beamline X12-C at the NSLS. © 1997 Wiley-Liss, Inc.     

The structure of Lactobacillus brevis surface layer reassembled on liposomes differs from native structure as revealed by SAXS

The reassembly of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 on positively charged liposomes was studied by small angle X-ray scattering (SAXS) and zeta potential measurements. SlpA was reassembled on unilamellar liposomes consisting of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-3-trimethylammonium-propane, prepared by extrusion through membranes with pore sizes of 50 nm and 100 nm. Similarly extruded samples without SlpA were used as a reference. The SlpA-containing samples showed clear diffraction peaks in their SAXS intensities. The lattice constants were calculated from the diffraction pattern and compared to those determined for SlpA on native cell wall fragments. Lattice constants for SlpA reassembled on liposomes (a = 9.29 nm, b = 8.03 nm, and γ = 84.9°) showed a marked change in the lattice constants b and γ when compared to those determined for SlpA on native cell wall fragments (a = 9.41 nm, b = 6.48 nm, and γ = 77.0°). The latter are in good agreement with values previously determined by electron microscopy. This indicates that the structure formed by SlpA is stable on the bacterial cell wall, but SlpA reassembles into a different structure on cationic liposomes. From the (10) reflection, the lower limit of crystallite size of SlpA on liposomes was determined to be 92 nm, corresponding to approximately ten aligned lattice planes.     

Purification,crystallization, and preliminary X-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis

The tRNA modifying enzyme tRNA–guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-aminomethyl-7-deazaguanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 Å, b = 65.1 Å, c = 71.9 Å, and β = 97.5°, and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 Å resolution.