Purification, crystallization and preliminary analysis of hemoglobin from rabbit (Oryctolagus cuniculus)

Hemoglobin (Hb) is a tetrameric protein, which contains four heme prosthetic groups, and each one is associated with a polypeptide chain. Herein, we report the rabbit hemoglobin which has intrinsically high oxygen affinity and possess highest sequence identity with human hemoglobin. The purified hemoglobin has been tried to crystallize in different crystallization conditions owing to its formation of various crystal systems. The rabbit Hb crystals were grown using PEG 3,350 as the precipitant at 18 degrees C. The crystals of rabbit Hb belongs to triclinic space group P1 with one molecule (alpha2beta2) in the asymmetric unit.     

Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection

For cryo-EM structural studies, we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the approximately 100nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.     

Purification and crystallization of the light harvesting LH1 complex from Rhodobacter sphaeroides.

The LH1 light harvesting complex has been purified from a mutant of the photosynthetic bacterium Rhodobacter sphaeroides which synthesizes LH1 as the sole pigment protein. Crystallization trials using polyethylene glycol as the precipitant in the presence of the detergent n-octyl glucoside have resulted in the formation of needle like crystals which diffract beyond 3.5 A and which are relatively resistant to radiation damage. X-ray photographs have established that the crystals belong to the tetragonal system and are probably in space group P4(2)2(1)2. Estimates of the crystal density indicate that the asymmetric unit of the crystals contains two oligomers each with an alpha 6 beta 6 stoichiometry.     

Factors Affecting Formation of Large Calcite Crystals (≥1 mm) in Bacillus subtilis 168 Biofilm

B4 is the most common medium used in general organomineralization studies and has been used to assay or to characterize mineral precipitation potential. In an exercise for the optimization of the laboratory conditions of crystal precipitation in vitro, we used Bacillus subtilis 168 as a type strain and its isogenic mutants. While literature is mainly focused on observing generic precipitation, we investigated the requirement to obtain large crystals (≥1 mm), which could be advantageous in wide-ranging implications for bioconsolidation of soil, sand, stone, and cementitious materials. Calcite crystals are visible on B4 agar plates within 7 days at 37°C after inoculum of B. subtilis 168 strain. In this study, we show that to form large crystals with a diameter ≥1 mm several conditions must be met: i) Reduced amount of B4 medium into the Petri plate improve crystal formation. 55 mm Petri plates contained only 4 ml of B4 agar medium reached a plateau in 6 days at 37°C. High moisture and presence of water condense would decrease crystal formation. ii) Inoculation of cells using a rod instead of a circular shaped spot. When the same number of B. subtilis cells was streaked, rod-shape biofilm significantly fostered crystal precipitation, while spot-shape prevented precipitation. iii) When more than one biofilm is present within the same plate, mutual interactions can affect precipitation in each biofilm. iv) Spherical nucleation sites are identified as initial step during the formation of large calcite crystal.     

Structural variability and new intermolecular interactions of Z-DNA in crystals of d(pCpGpCpGpCpG).

We have determined the single crystal x-ray structure of the synthetic DNA hexamer d(pCpGpCpGpCpG) in two different crystal forms. The hexamer pCGCGCG has the Z-DNA conformation and in both cases the asymmetric unit contains more than one Z-DNA duplex. Crystals belong to the space group C222(1) with a = 69.73, b = 52.63, and c = 26.21 A, and to the space group P2(1) with a = 49.87, b = 41.26, c = 21.91 A, and gamma = 97.12 degrees. Both crystals show new crystal packing modes. The molecules also show striking new features when compared with previously determined Z-DNA structures: 1) the bases in one duplex have a large inclination with respect to the helical axis, which alters the overall shape of the molecule. 2) Some cytosine nitrogens interact by hydrogen bonding with phosphates in neighbor molecules. Similar base-phosphate interactions had been previously detected in some B-DNA crystals. 3) Basepair stacking between the ends of neighbor molecules is variable and no helical continuity is maintained between contiguous hexamer duplexes.     

The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals

Magnetospirillum gryphiswaldense and related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe₃O₄) that cause the cells to orient in magnetic fields. The characteristic sizes, morphologies, and patterns of alignment of magnetite crystals are controlled by vesicles formed of the magnetosome membrane (MM), which contains a number of specific proteins whose precise roles in magnetosome formation have remained largely elusive. Here, we report on a functional analysis of the small hydrophobic MamGFDC proteins, which altogether account for nearly 35% of all proteins associated with the MM. Although their high levels of abundance and conservation among magnetotactic bacteria had suggested a major role in magnetosome formation, we found that the MamGFDC proteins are not essential for biomineralization, as the deletion of neither mamC, encoding the most abundant magnetosome protein, nor the entire mamGFDC operon abolished the formation of magnetite crystals. However, cells lacking mamGFDC produced crystals that were only 75% of the wild-type size and were less regular than wild-type crystals with respect to morphology and chain-like organization. The inhibition of crystal formation could not be eliminated by increased iron concentrations. The growth of mutant crystals apparently was not spatially constrained by the sizes of MM vesicles, as cells lacking mamGFDC formed vesicles with sizes and shapes nearly identical to those formed by wild-type cells. However, the formation of wild-type-size magnetite crystals could be gradually restored by in-trans complementation with one, two, and three genes of the mamGFDC operon, regardless of the combination, whereas the expression of all four genes resulted in crystals exceeding the wild-type size. Our data suggest that the MamGFDC proteins have partially redundant functions and, in a cumulative manner, control the growth of magnetite crystals by an as-yet-unknown mechanism.     

Studies on shell formation. VII. The submicroscopic structure of the shell of the oyster Crassostrea virginica

The submicroscopic structure of the growing surface of the shell of the oyster, Crassostrea virginica, was studied by means of shadowed replicas. The outer edge of the prismatic region consists of a fine grained matrix enclosing crystals, the surfaces of which show a finely pebbled structure. Crystal size varies continously from 0.01 micro to 8 micro. The matrix surface shows no evidence of fibrous structure. The outer portions of the prismatic region exhibit a tile-like arrangement of large crystals separated by granular matrix 0.02 to 0.08 micro in thickness. The exposed crystal surfaces have indentations of varying form which appear as roughly parallel grooves spaced at intervals of approximately 0.3 micro. The final form of this region is believed to result from the random distribution of crystal seeds, which grow without orientation and through coalescence and growth come into contact, producing polygonal areas. The crystal arrangement of the nacreous region is one of overlapping rows of crystals in side to side contact, and with one end of each crystal free, permitting continued increase in length. Crystal angles and plane indices are presented.     

Freezing of tissue-limits for the autoradiographic localization of diffusible substances

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.     

Improved production of the insecticidal CryIVD protein in Bacillus thuringiensis using cryIA(c) promoters to express the gene for an associated 20-kDa protein

Previous studies have shown that a 20-kDa protein enhances production of the insecticidal CytA and CryIVA proteins of Bacillus thuringiensis in Escherichia coli as well as CytA production and crystal formation in B. thuringiensis. To determine whether the 20-kDa protein could enhance CryIVD production, an expression vector was constructed with the 20-kDa open-reading frame under control of cryIA(c) promoters and the cryIVD gene under control of its own promoter. Acrystalliferous cells of B. thuringiensis transformed with this plasmid, designated pWF53, produced large bitrapezoidal CryIVD crystals that averaged 1.3 × 0.92 × 0.31 m, approximately fivefold larger in volume than wild-type CryIVD crystals, and 1.7 fold the volume of crystals produced using the cryIV D operon, which contains the cryIV D gene and the gene for the 20-kDa protein. These results demonstrate that the 20-kDa protein significantly improves net synthesis of CryIVD and promotes CryIVD crystal formation. Improved production of proteins as diverse as CryIVD and CytA by the 20-kDa protein indicates this protein may be useful in facilitating the production of other proteins.     

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.     

苏云金芽孢杆菌工程菌伴孢晶体的形态发生

苏云金芽孢杆菌在有帮助蛋白存在的情况下杀虫晶体蛋白获得了超量表达。通过透射电镜观察了Cry1Ac超量表达工程菌伴孢晶体的形态发生以及不同芽孢发育时期的晶体形态变化。结果表明,该工程菌的伴孢晶体在细胞不对称分裂的隔膜形成前就已出现,而且晶体发生的部位与芽孢无关。但晶体在形成初期往往靠近母细胞膜。观察结果还表明,大量表达的晶体蛋白不能马上参与到晶体合成,晶体形成的最佳时期是芽孢皮层形成期。母细胞大量液泡的产生与消失可能与晶体形成有关。此外,在超量表达工程菌中,Cry1Ac蛋白能在一个细胞内形成多个伴孢晶体,这在天然菌株中是罕见的。     

莫桑鼻给非鲫滤泡闭锁中液晶形成的冰冻蚀刻研究

本文运用电镜的冰冻蚀刻术研究了莫桑鼻给非鲫滤泡闭锁中液晶形成的过程.结果表明,卵巢内的颗粒细胞吞噬大量的卵黄物质,消化后形成同心圆片层体,这是一种类脂加水以及镶嵌少量的蛋白质的溶致液晶态;细胞内的酶类参与液晶的形成;同时讨论了生物体内相变及液晶态存在的意义.     

莫桑鼻给非鲫滤泡闭锁中液晶形成的冰冻蚀刻研究

本文运用电镜的冰冻蚀刻术研究了莫桑鼻给非鲫滤泡闭锁中液晶形成的过程.结果表明,卵巢内的颗粒细胞吞噬大量的卵黄物质,消化后形成同心圆片层体,这是一种类脂加水以及镶嵌少量的蛋白质的溶致液晶态;细胞内的酶类参与液晶的形成;同时讨论了生物体内相变及液晶态存在的意义.     

Helical crystals of sickle cell hemoglobin

Thin ribbon-like crystals are intermediates in the formation of large crystals of deoxyhemoglobin S from many individual fibers. The thin crystals show foldedover regions when observed by electron microscopy. Some crystals are sufficiently long to have several folds each separated by a distance of about 4.4 μm, suggesting that the crystals are helical in solution. The thickness of the crystals varies from 500 to 900 Å as shown by heavy-metal shadowing and by measurements of the thickness at the crossover point where an edge-on view of the crystal is obtained.     

Two-dimensional crystallization of Ca-ATPase by detergent removal.

By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain.     

Development of secretory cells and crystal cells in Eichhornia crassipes ramet shoot apex

The distribution and development of secretory cells and crystal cells in young shoot apexes of water hyacinth were investigated through morphological and cytological analysis. The density of secretory cells and crystal cells were high in parenchyma tissues around the vascular bundles of shoot apexes. Three developmental stages of the secretory cells can be distinguished under transmission electron microscopy. Firstly, a large number of electron-dense vesicles formed in the cytoplasm, then fused with the tonoplast and released into the vacuole in the form of electron-dense droplets. As these droplets fused together, a large mass of dark material completely filled the vacuole. To this end, a secretion storage vacuole (SSV) formed. Secondly, an active secretion stage accompanied with degradation of the large electron-dense masses through an ill-defined autophagic process at periphery and in the limited internal regions of the SSV. Finally, after most storage substances were withdrawn, the materials remaining in the spent SSV consisted of an electron-dense network structure. The distribution and development of crystal cells in shoot apical tissue of water hyacinth were also studied by light and electron microscopy. Crystals initially formed at one site in the vacuole, where tube-like membrane structures formed crystal chambers. The chamber enlarged as the crystal grew in bidirectional manner and formed needle-shaped raphides. Most of these crystals finally occurred as raphide bundles, and the others appeared as block-like rhombohedral crystals in the vacuole. These results suggest that the formation of both secretory cells and crystal cells are involved in the metamorphosis of vacuoles and a role for vacuoles in water hyacinth rapid growth and tolerance.     

Studies on Shell Formation : VII. The Submicroscopic Structure of the Shell of the Oyster Crassostrea virginica

The submicroscopic structure of the growing surface of the shell of the oyster, Crassostrea virginica, was studied by means of shadowed replicas. The outer edge of the prismatic region consists of a fine grained matrix enclosing crystals, the surfaces of which show a finely pebbled structure. Crystal size varies continously from 0.01 µ to 8 µ. The matrix surface shows no evidence of fibrous structure. The outer portions of the prismatic region exhibit a tile-like arrangement of large crystals separated by granular matrix 0.02 to 0.08 µ in thickness. The exposed crystal surfaces have indentations of varying form which appear as roughly parallel grooves spaced at intervals of approximately 0.3 µ. The final form of this region is believed to result from the random distribution of crystal seeds, which grow without orientation and through coalescence and growth come into contact, producing polygonal areas. The crystal arrangement of the nacreous region is one of overlapping rows of crystals in side to side contact, and with one end of each crystal free, permitting continued increase in length. Crystal angles and plane indices are presented.     

Converting bacteriophage for sporulation and crystal formation in Bacillus thuringiensis.

Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted anoligosporogenic (sporulation frequency, 10(-8), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the ho-t used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10(-6) to 10(-5). Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis.     

Ca2+ as developmental signal in the formation of Ca-oxalate crystal spacing patterns during leaf development inCarya ovata

Changes in the spacing patterns of Ca-oxalate crystals during enlargement ofCarya ovata Mill. leaves were quantified by computerized image-analysis. Single Ca-oxalate crystals form in the vacuoles of young mesophyll cells transformed into crystal cells Crystals are very small in newly induced crystal cells and increase in size throughout leaf development. Crystal patterns thus reflect both induction and relative age of crystal cells. Shortly after the emergence of young leaves from the bud, very small crystals are formed in the mesophyll at high density. As leaves expand, these crystals grow larger and become separated by increasing distances. New small crystals appear in the gaps between the older, larger crystals. Later crystal patterns consist of widely spaced, larger crystals only. Finally, clusters of small crystals are formed again in the gaps between large crystals. No crystals were observed in young leaves expanding in a moist chamber, but large numbers of crystal cells were induced experimentally in sections of immature leaves floating on 4 mM Ca-acetate. The observations support the following mechanism of crystal-pattern formation: Ca²⁺ carried into leaves with the transpiration stream acts as the developmental signal inducing transdifferentiation of a few mesophyll cells into crystal cells when apoplastic [Ca²⁺] rises. Crystal cells precipitate absorbed Ca²⁺ as oxalate and, acting as Ca²⁺ sinks, inhibit crystal-cell induction in their vicinity by depleting apoplastic Ca²⁺. This prevents close spacing of crystal cells. New crystal cells form in the gaps between the depletion zones of older crystal cells when these move apart during leaf expansion. Later changes in crystal patterns result from increasing sink strength of crystal cells, lowered inducibility of mesophyll cells, and increased Ca²⁺ influx into leaves during intensive transpiration. Throughout leaf development, spacing of crystal cells permits rapid secretion of apoplastic Ca²⁺ as Ca-oxalate. Dedicated to Professor Erwin Bünning, University of Tübingen, Germany, who pioneered the analysis of spacing patterns