New Crystal Forms of the Motile Major Sperm Protein (MSP) ofAscaris suum

We have obtained two new crystal forms of theAscarismajor sperm protein (MSP) that mediates amoeboid cell motility in nematode sperm. We obtained crystals with C2 symmetry from bacterially expressed α-MSP witha= 216.5 Å,b= 38.6 Å,c= 32.5 Å, γ = 93.1° and also crystals with P2₁symmetry from native β-MSP witha= 63.1 Å,b= 91.7 Å,c= 72.5 Å, γ = 91.3°. A full native data set has been collected for each crystal form using synchrotron radiation. Both crystal forms diffract to 2 Å and are suitable for high-resolution structural investigation.     

Type IIβ Regulatory Subunit of cAMP-Dependent Protein Kinase: Purification Strategies to Optimize Crystallization

To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RIIβ isoform and five RIIβ deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RIIβ deletion mutant (Δ1–111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 Å resolution were collected at 29°C using synchrotron radiation on a single crystal measuring 0.2 × 0.3 × 1.2 mm³. Data were 99% complete. The hexagonal crystal belonged to space group P6₍₁₎ or P6₍₅₎ with unit cell dimensions a = b = 161.62 Å and c = 39.66 Å.     

The ultrastructure of the larval malpighian tubules of a saline-water mosquito

The larval Malpighian tubules of the saline-water mosquito Aedes taeniorhynchus were examined using light and electron microscopy. The tubules contain two cell types: primary cells and stellate cells. Primary cells are characterized by their size (70 μm × 70 μm × 10 μm) and an abundance of intracellular membranebound crystals. Two types of microvilli are found on the luminal surface of the primary cells: (1) small microvilli containing core microfilaments and extensions of endoplasmic reticulum, and (2) larger microvilli (≈3 μm in length) which in addition to the above components contain a mitochondrion along their entire length. Both microvillar types have abundant knobs lining the cytoplasmic surface of the microvillar membrane. These knobs, which are often found in insect ion transporting tissues, have been termed ‘portasomes’ by Harvey (1980). The possible role of these structures in ion transport and mitochondrial positioning is discussed. The stellate cells are much smaller than the primary cells, and lack intracellular crystals. Their microvilli are smaller as well (≈0.6 μm in length) and contain no endoplasmic reticulum. mitochondria or knobs. The cells types found in the saline-water mosquito larva, Aedes taeniorhynchus, are identical to those found in Aedes aegypti, indicating that the unique capacity of saline-water mosquito larvae to transport Mg²⁺ and SO₄|post|staggered|2− is not associated with the presence of an additional cell type.     

Three-Dimensional Structure of the Porcine Gastric H,K-ATPase from Negatively Stained Crystals

A low-resolution three-dimensional model of membrane-bound H,K-ATPase from pig gastric mucosa has been reconstructed by electron microscopy and image processing of two-dimensional crystals in negative stain. The crystal formation is induced by magnesium and vanadate, which stabilize the E₂conformation of the enzyme. The unit cell, with a size ofa=b= 123 Å, γ = 90°, has tetragonal p4 symmetry. There are four separate αβ protomers within each unit cell. The high-contrast region is limited to the cytoplasmic part of the protein. The total volume of the observed asymmetric protein domain corresponds to a molecular mass of 80–90 kDa. It consists mainly of a large pear-shaped domain measuring 60 × 45 Ų, with a height of 50 Å as measured perpendicular to the membrane plane. A small stalk segment, 20 Å in length, forms a connection to the transmembrane region.     

Annual fish oogenesis : I. Differentiation of the mature oocyte and formation of the primary envelope

The chorion surface in the eggs of the annual fishes Cynolebias melanotaenia and C. ladigesi contains an elaborate, three-dimensional species-specific pattern. Two concentric layers form the chorion. The pattern resides in the outer layer, the secondary envelope. It consists of closely packed tubules about 250 Å in diameter. A coat of electron dense “fuzzy” material increases this to 475 Å. The inner layer, the primary envelope, of uniformly low electron density possesses no obvious substructure. Oogenesis is divided into six stages. The oocyte increases in size from 10–20 μm in Stage 1 to 250 μm in Stage 3, 600 μm in Stage 4, and attains maximal size of 900 μm by Stage 6. Massive inclusions of protein and lipid yolk accumulate during Stages 4 and 5. Zone 1, one of the three zones of the primary envelope, first appears late in Stage 2. During Stage 3, Zone 1 is completed and Zone 2 appears between the oocyte surface and Zone 1. The oocyte cytoplasm increases in complexity. Material similar to Zone 1 (light, fibrillar) and Zone 2 (dark, compact) is present in the RER, Golgi, derivative vesicles, and apical pits. Micropyle formation also commences. The oocyte secretes Zone 3 during Stage 4 as thin filaments which consolidate into a highly ordered, transitional structure composed of tangentially oriented bundles of interwoven filaments. These partially fuse during Stage 5 except for fenestrations through which oocyte and follicle cell microvilli pass. Complete fusion during Stage 6 produces a continuous layer. Follicle cells retain an unspecialized structure from Stages 1 through 4. Secondary envelope material accumulates in the RER of the follicle cells during Stage 5. It is secreted and deposited during Stage 6.     

Crystallization and Preliminary X-Ray Diffraction Analysis of the 190-Å-Long Coiled-Coil Dimerization Domain of the Actin-Bundling Protein Cortexillin I fromDictyostelium discoideum

We have crystallized the ≈190-Å-long parallel two-stranded coiled-coil oligomerization domain of the actin-bundling protein cortexillin I fromDictyostelium discoideum. The orthorhombic crystals belong to the space group C222₁with unit cell dimensions ofa= 71.3 Å,b= 127.8 Å, andc= 91.6 Å. As both native and selenomethionine-substituted protein crystals diffract to 3.0 and 2.85 Å resolution, respectively, using synchrotron radiation, they are suitable for the first high-resolution structural analysis of a two-stranded coiled coil comprising more than six heptad repeats. Moreover, because the polypeptide chain fragment contains a recently identified two-heptad-repeat long sequence that is indispensable for the assembly of the cortexillin I coiled-coil oligomerization domain, its high-resolution structure should enable us to extend our knowledge on the molecular mechanisms underlaying coiled-coil formation and to establish the precise manner in which the two “trigger” sequences interact with one another in the dimer.     

Structural investigations of lipid, polypeptide and protein multilayers

Langmuir-Blodgett multilayers of lipids, polypeptides and proteins have been examined by X-ray diffraction and infrared spectroscopic methods. The complex polymorphism exhibited by multilayers of glycerides and various phospholipids of different chain length mirror those shown in other three-dimensional structures and suggest that multilayers of lipids can be considered as oriented “crystals”. Both the α and β types of hdyrocarbon chain packing are adopted by different classes of lipids in multilayers.Stable multilayers of the synthetic polypeptide poly-γ-benzyl-l-glutamate consist of α-helical rods stacked in an hexagonal array with a rod axis separation of 14.2 Å. Poly-γ-methyl-l-glutamate behaves similarly but little structural information could be derived from potentially non-helical or sheet-like structures formed by other homopolypeptides. The observation of a single, invariant diffraction line at 9.3 Å for multilayers of a number of water-soluble proteins is consistent with the occurrence of extensive structural reorganization (uncoiling, denaturation) at the air-water interface.     

Two-Dimensional Crystallization of Brush Border Myosin I

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22₁2₁or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 Å and show that the asymmetric unit is the same in both crystal types.     

Calcium Oxalate Crystals: An Integral Component of the Sclerotinia sclerotiorum/Brassica carinata Pathosystem

Oxalic acid is an important virulence factor for disease caused by the fungal necrotrophic pathogen Sclerotinia sclerotiorum, yet calcium oxalate (CaOx) crystals have not been widely reported. B. carinata stems were infected with S. sclerotiorum and observed using light microscopy. Six hours post inoculation (hpi), CaOx crystals were evident on 46% of stem sections and by 72 hpi on 100%, demonstrating that the secretion of oxalic acid by S. sclerotiorum commences before hyphal penetration. This is the first time CaOx crystals have been reported on B. carinata infected with S. sclerotiorum. The shape of crystals varied as infection progressed. Long tetragonal rods were dominant 12 hpi (68% of crystal-containing samples), but by 72 hpi, 50% of stems displayed bipyramidal crystals, and only 23% had long rods. Scanning electron microscopy from 24 hpi revealed CaOx crystals in all samples, ranging from tiny irregular crystals (< 0.5 μm) to large (up to 40 μm) highly organized arrangements. Crystal morphology encompassed various forms, including tetragonal prisms, oval plates, crystal sand, and druses. Large conglomerates of CaOx crystals were observed in the hyphal mass 72 hpi and these are proposed as a strategy of the fungus to hold and detoxify Ca²⁺ions. The range of crystal morphologies suggests that S. sclerotiorum growth and infection controls the form taken by CaOx crystals.     

Crystal Structure Determination of FtsZ fromMethanococcus jannaschii

FtsZ is the polymer-forming protein of bacterial cell division. It is part of a ring in the middle of the dividing cell that is required for constriction of cell membrane and cell envelope to yield two daughter cells. FtsZ is a GTPase and is the only bacterial protein showing significant sequence homology to the eukaryotic tubulins. FtsZ can polymerize into tubes, sheets, and ringsin vitroand is ubiquitous in eubacteria and archaea. Full-length FtsZ1 fromMethanococcus jannaschiihas been over expressed inEscherichia coli,employing the hyperthermophilic properties of the protein. Crystals grown from PEG400 and ethanol belong to spacegroup I2₁3 witha=b=c= 159.1 Å. Isomorphous replacement using one Hg derivative yielded a interpretable electron density map at 4 Å resolution. The structure for residues 23–356 and one GDP has been refined to anR_freeof 0.28 (R_f = 0.20) at 2.8 Å resolution. FtsZ consists of two domains with a connecting core helix. The N-terminal domain and the core helix contain all residues involved in nucleotide binding and resemble the fold of dinucleotide-binding proteins. The structures of tubulin and FtsZ show striking similarity; together with the functional similarities, this provides a strong indication that FtsZ is a true homolog of tubulin.     

Structure of the tubulin dimer in zinc-induced sheets

The structure of tubulin has been studied in projection by minimum beam electron microscopy and image processing of negatively stained zinc-induced sheets. The reconstructed images include data to 15 Å resolution.We report here a clear and reproducible 82 Å repeat arising from the arrangement of heterodimers in sheet aggregates of tubulin. This repeat is only observed in diffraction patterns from images recorded by minimum beam methods (10 to 20 e/Ų) and arises from small, but consistent, structural differences between two similar subunits believed to represent the two chemical species of tubulin monomer (M_r, 55,000). At higher electron doses (100 to 200 e/Ų), the additional information is lost or very much reduced, and only a repeat of 41 Å is observed, owing to the loss of distinction between monomers in the tubulin heterodimer.The sheets are composed of 49 Å wide, polar protofilaments, similar to those observed in microtubules; however, the interprotofilament packing is completely different in the two structures. In these sheets, adjacent protofilaments point and face in opposite directions; i.e. they are related by dyad-screw axes normal to the protofilament axes and in the plane of the sheet. Thus, the zinc-induced sheets are crystals of space group P2₁, with cell dimensions of about 97 Å × 82 Å, containing one tubulin heterodimer per asymmetric unit.Reconstructed images of four individual sheets, and their average, show the arrangement and shapes of the two heterodimers contained in each unit cell. The structure and packing of heterodimers in sheets are compared to those in opened out microtubules where all protofilaments point and face in the same direction.     

Projection maps of three members of the KdgM outer membrane protein family

We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7 Å. All three proteins exhibit a similar putative β-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using β-barrel specific prediction algorithms. The predicted transmembrane β-barrels have a high similarity in the arrangement of the putative β-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.     

How is the flagellar length of mature sperm determined? : II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 μm, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 μm, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.     

The structures of bis(1H,5H-S-methylisothiocarbonohydrazidium) di-μ-chloro-octachlorodibismuthate(III) tetrahydrate and tris(1H-S-methylisothiocarbonohydrazidium) esachlorobismuthate(III)

The structures of bis(1H⁺,5H⁺-S-methylisothiocarbonohydrazidium) di-μ-chlorooctachlorodibismuthate(III) tetrahydrate: (C₂H₁₀N₄S)₂(Bi₂Cl₁₀)· 4H₂O (compound [I]) and of tris(1H⁺-S-methylisothiocarbonohydrazidium) esachlorobismuthate(III): (C₂H₉N₄S)₃(BiCl_5.67I_0.33) (compound [II]) were determined from single crystal X-ray diffractometer data. Both compounds crystallize as triclinic (P ); crystals [I] with Z = 1 formula unit in a cell of constants: A = 10.621(3), B = 9.989(5), C = 7.439(3) Å, α = 88.31(2), β = 84.51(2), γ = 68.88(2)°, final R = 0.0427 for 2229 unique reflections with I 2σ(I); crystals [II] with Z = 2 and cell dimensions: A = 14.109(4), B = 12.209(9), C = 8.206(7) Å, α = 103.54(3), β = 104.95(2), γ = 81.96(2)°, final R = 0.0411 for 3637 unique reflections (1 2σ(I)). The structure of [I] is built up of diprotonated organic cations, water molecules and dinuclear centrosymmetric [Bi₂Cl₁₀]⁴⁻ anions held together by N-HCl, N-HO, O-HCl hydrogen bonds and Van der Waals interactions. The [Bi₂Cl₁₀]⁴⁻ complex consists of two edge-sharing octahedra in which three pairs of bonds of similar length are observed (Bi-Cl_av = 2.602(5), 2.712(4), 2.855(5) Å). The structure of [II] consists of monoprotonated cations and [BiCl_5.67I_0.33]³⁻ anions held together by a tridimensional network of hydrogen bonds. Each bismuth atom is octahedrally surrounded by six chlorine atoms, one of which is statistically substituted by a iodine atom.     

Optimizing the crystal size and habit of β-sitosterol in suspension

The aim of this work was to survey how processing parameters affect the crystal growth of β-sitosterol in suspension. The process variables studied were the cooling temperature, stirring time and stirring rate during recrystallization. In addition, we investigated the effect a commonly used surfactant, polysorbate 80, has on crystal size distribution and the polymorphic form. This study describes the optimization of the crystallization process, with the object of preparing crystals as small as possible. Particle size distribution and habit were analyzed using optical microscopy, and the crystal structure was analyzed using X-ray diffractometry. The cooling temperature had a remarkable influence on the crystal size. Crystals with a median crystal length of ≈23 μm were achieved with a low cooling temperature (<10°C); however, a fairly large number of crystals over 50 μm appeared. Higher cooling temperatures (>30°C) caused notable crystal growth both in length and width. Rapid (250 rpm), continuous stirring until the suspensions had cooled to room temperature created small, less than 50 μm long (median <20 μm), needle-shaped crystals. The addition of surfactant slightly reduced the size of the initially large crystals. Both hemihydrate and monohydrate crystal forms occurred throughout, regardless of the processing parameters. By using an optimized process, it was possible to obtain a microcrystalline suspension, with a smooth texture.     

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.     

Dynamics of Iron Uptake and Fe3O4 Biomineralization during Aerobic and Microaerobic Growth of Magnetospirillum gryphiswaldense

Iron uptake and magnetite (Fe₃O₄) crystal formation could be studied in the microaerophilic magnetic bacterium Magnetospirillum gryphiswaldense by using a radioactive tracer method for iron transport and a differential light-scattering technique for magnetism. Magnetite formation occurred only in a narrow range of low oxygen concentration, i.e., 2 to 7 μM O₂ at 30°C. Magnetic cells stored up to 2% iron as magnetite crystals in intracytoplasmic vesicles. This extraordinary uptake of iron was coupled tightly to the biomineralization of up to 60 magnetite crystals with diameters of 42 to 45 nm.     

Metagenomic analysis of size-fractionated picoplankton in a marine oxygen minimum zone

Marine oxygen minimum zones (OMZs) support diverse microbial communities with roles in major elemental cycles. It is unclear how the taxonomic composition and metabolism of OMZ microorganisms vary between particle-associated and free-living size fractions. We used amplicon (16S rRNA gene) and shotgun metagenome sequencing to compare microbial communities from large (>1.6 μm) and small (0.2–1.6 μm) filter size fractions along a depth gradient in the OMZ off Chile. Despite steep vertical redox gradients, size fraction was a significantly stronger predictor of community composition compared to depth. Phylogenetic diversity showed contrasting patterns, decreasing towards the anoxic OMZ core in the small size fraction, but exhibiting maximal values at these depths within the larger size fraction. Fraction-specific distributions were evident for key OMZ taxa, including anammox planctomycetes, whose coding sequences were enriched up to threefold in the 0.2–1.6 μm community. Functional gene composition also differed between fractions, with the >1.6 μm community significantly enriched in genes mediating social interactions, including motility, adhesion, cell-to-cell transfer, antibiotic resistance and mobile element activity. Prokaryotic transposase genes were three to six fold more abundant in this fraction, comprising up to 2% of protein-coding sequences, suggesting that particle surfaces may act as hotbeds for transposition-based genome changes in marine microbes. Genes for nitric and nitrous oxide reduction were also more abundant (three to seven fold) in the larger size fraction, suggesting microniche partitioning of key denitrification steps. These results highlight an important role for surface attachment in shaping community metabolic potential and genome content in OMZ microorganisms.     

New and unusual forms of calcium oxalate raphide crystals in the plant kingdom

Calcium oxalate crystals in higher plants occur in five major forms namely raphides, styloids, prisms, druses and crystal sand. The form, shape and occurrence of calcium oxalate crystals in plants are species- and tissue-specific, hence the presence or absence of a particular type of crystal can be used as a taxonomic character. So far, four different types of needle-like raphide crystals have been reported in plants. The present work describes two new and unusual forms of raphide crystals from the tubers of Dioscorea polystachya—six-sided needles with pointed ends (Type V) and four-sided needles with beveled ends (Type VI). Both of these new types of needles are distinct from the other four types by each having a surrounding membrane that envelopes a bundle of 10–20 closely packed thin crystalline sheets. The previously known four types of needles have solid or homogenous crystalline material, surrounded by a membrane or lamellate sheath called a crystal chamber. Only the Type VI crystals have beveled ends and the needles of the other five types have pointed ends.