Habits of Magnetosome Crystals in Coccoid Magnetotactic Bacteria

High-resolution transmission electron microscopy and electron holography were used to study the habits of exceptionally large magnetite crystals in coccoid magnetotactic bacteria. In addition to the crystal habits, the crystallographic positioning of successive crystals in the magnetosome chain appears to be under strict biological control.     

The Occurrence of Biogenic Calcian Struvite, (Mg, Ca)NH4PO4.6H2O, as Intracellular Crystals in Paramecium

ABSTRACT. Intracellular crystals are conspicuous refractile "inclusion bodies" commonly found in many protozoans, but very few have been identified mineralogically. We have isolated crystals from axenically grown mass cultures of Paramecium tetraurelia , and purified them using differential centrifugation. The crystals' structure and chemistry were analyzed using x-ray powder diffraction and energy-dispersive electron microprobe techniques. The morphology was studied by means of scanning electron microscopy. The crystals were identified as the orthorhombic mineral, calcian struvite, (Mg, Ca)NH₄PO₄.6H₂O. Struvite from P. tetraurelia exhibited a variety of crystal habits, including hemimorphic forms, epitactic overgrowths and several types of twins. A linear correlation between computed hydration number and Mg content suggests that the crystal composition may reflect the range of conditions under which struvite nucleation and growth occur. The mineral struvite also occurs in association with guano and other rich organic products, and can be biologically induced to precipitate extracellularly. Extracellular struvite has been well characterized in pathogenic calculi (kidney stones) of humans and cats, where precipitation is enhanced by bacterial urease activity that produces ammonia in the urinary tract. This is the first study demonstrating that struvite is also biologically controlled to form as an intracellular mineral. These crystals may have formed within lipid-rich, membrane-bound vesicles in Paramecium .     

ASSOCIATION OF FOUR DIFFERENT CALCIUM CRYSTALS IN THE ANTHER CONNECTIVE TISSUE AND HYPODERMAL STOMIUM OF CAPSICUM ANNUUM (SOLANACEAE) DURING MICROSPOROGENESIS

The anther connective tissue and hypodermal stomium between adjacent locules in the anthers of Capsicum annuum L. (Solanaceae) are the sites of formation of calcium salt crystals with four different habits. The spatial and temporal associations of these crystals and the idioblastic cells in which they form indicate that crystal sand occurs earliest in anther development near the single vascular strand, followed by spherulites and prismatic crystals farther out in the connective tissue, and finally druses occur in the hypodermal stomium. Both the druses and the crystal sand crystals are encased in crystal chambers and are associated with distinct membranes, whereas the spherulites and prismatic crystals are not bounded by any apparent membranes but they are surrounded by dense material that is rich in calcium and stains positively for polysaccharides and proteins. Quite often spherulites and prismatic crystals are observed within a single cell in contact with each other. X-ray diffraction of crystal preparations containing all four crystal habits and X-ray elemental analyses of single crystals, as well as visual observations and acid treatments, suggest that all four crystal habits consist of calcium oxalate. The hypodermal stomium and adjacent connective tissue degenerate at the pollen stage causing adjacent locules to fuse. Shortly afterward, each stomium epidermis splits open along the length of the anther releasing the pollen. It is suggested that the crystal idioblasts are involved in this process, possibly by a temporally orchestrated sequestration of calcium from both the cell cytoplasm and cell wall.     

Protein and virus crystal growth on international microgravity laboratory-2.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.     

Atomic force microscopy study of tooth surfaces

Atomic force microscopy (AFM) was used to study tooth surfaces in order to compare the pattern of particle distribution in the outermost layer of the tooth surfaces. Human teeth and teeth from a rodent (Golden hamster), from a fish (piranha), and from a grazing mollusk (chiton) with distinct feeding habits were analyzed in terms of particle arrangement, packing, and size distribution. Scanning electron microscopy and transmission electron microscopy were used for comparison. It was found that AFM gives high-contrast, high-resolution images and is an important tool as a source of complementary and/or new structural information. All teeth were cleaned and some were etched with acidic solutions before analysis. It was observed that human enamel (permanent teeth) presents particles tightly packed in the outer surface, whereas enamel from the hamster (continuously growing teeth) shows particles of less dense packing. The piranha teeth have a thin cuticle covering the long apatite crystals of the underlying enameloid. This cuticle has a rough surface of particles that have a globular appearance after the brief acidic treatment. The similar appearance of the in vivo naturally etched tooth surface suggests that the pattern of globule distribution may be due to the presence of an organic material. Elemental analysis of this cuticle indicated that calcium, phosphorus, and iron are the main components of the structure while electron microdiffraction of pulverized cuticle particles showed a pattern consistent with hydroxyapatite. The chiton mineralized tooth cusp had a smooth surface in an unabraded region and a very rough structure with the magnetite crystals (already known to make part of the structure) protruding from the surface. It was concluded that the structures analyzed are optimized for efficiency in feeding mechanism and life span of the teeth.     

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.     

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.     

Ultrastructure of the bacterial spores and crystals of various Bacillus thuringiensis serotypes

Spores and crystals of various Bacillus thuringiensis serotypes were studied by electron microscopy. The serotypes were shown to differ in the fine structure of the exosporium and crystals. The packing parameters of morphological subunits were determined by analysing electron photomicrographs by the technique of optical diffraction and filtration. Some of the strains are characterised by hypersynthesis of surface spore structures. The individual morphological properties of crystals can be used for their differentiation.     

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules.

Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free biotin-binding sites on the surface of the two-dimensional crystals facing the aqueous solution. The free biotin-binding sites could be specifically labeled with biotinylated ferritin. The streptavidin/biotinylated lipid system may provide a general approach for the formation of two-dimensional crystals of biotinylated macromolecules.     

Electron diffraction from single crystals of DNA.

Crystals of DNA have been grown in a form suitable for study by electron diffraction and electron microscopy. Preliminary electron diffraction patterns of any type that have been obtained from single crystals of highly polymerized DNA. The patterns, obtained from frozen, hydrated crystals with the beam approximately parallel to the DNA strand axis, show a hexagonal geometrical arrangement with a (1,0) Bragg spacing of 23.1 A.     

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography.

Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals.     

Using focus ion beam to prepare crystal lamella for electron diffraction

Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the electron crystallographic method on large protein crystals with micrometer or larger sizes. Here, we used the focused ion beam (FIB) equipped on the scanning electron microscope (SEM) to mill a large crystal to thin lamella. The influences of the milling on the crystal lamella were observed and investigated, including radiation damage on the crystal surface during the FIB imaging, deformation of the thin crystal lamella, and variation in the diffraction intensities under electron radiation. These observations provide important information to optimize the FIB milling, and hence is important to obtain high-quality crystal samples for routine structure determination of protein crystals using the electron cryo-microscope.     

A STUDY OF METASTATIC RENAL CALCIFICATION AT THE CELLULAR LEVEL

Experimental metastatic calcification in the proximal convoluted tubules of rat kidney, produced by large doses of vitamin D, has been studied with a variety of techniques. These techniques include the examination of thin sections of Araldite-embedded material under the electron microscope, selected area electron diffraction, and several histochemical methods. Two types of mineral are found in relation to the proximal convoluted tubule. The first form consists of aggregates of elongated crystals within cytoplasmic vacuoles of the proximal tubular cells. The dimensions of these crystals are consistent with those of hydroxyapatite. The other type of mineral deposit is found in and adjacent to the extracellular phase of the basal infoldings of these tubules. The latter deposits are made up of smaller crystals arranged in layers. These crystals could not be definitely identified by means of selected area electron diffraction. The observations are discussed in relation to calcium transport by the proximal convoluted tubule and also in terms of mechanisms of pathological calcification.     

Low dose electron microscopy of the crotoxin complex thin crystal

The crotoxin complex from Crotalus d. terrificus rattlesnake venom was crystallized in the form of thin platelets. These crystals were prepared by the glucose embedding technique and examined by low dose electron microscopy. Electron diffraction patterns and images have been recorded to 2.2 and 4.5 A, respectively. By a combination of electron and X-ray diffraction techniques, the space group of this crystal was determined to be P4(2)22 with eight crotoxin complex molecules in one unit cell with dimensions of 38.8 A x 38.8 A x 256.8 A. The Patterson maps and the symmetry reliability factors calculated from the electron diffraction intensities clearly showed the existence of three types of electron diffraction patterns in different crystals. The phases in the computer-calculated transform of the low dose images also show the variation in symmetry among crystals. These phenomena are explained by the presence of crystals consisting of one-half, three-quarter and one unit cell in thickness. The interpretation of the computer reconstructed two-dimensional density map was limited, partly because of the similarity in density between the protein and the embedding glucose and partly because of the non-uniqueness in relating projected structure to the three-dimensional structure.     

7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Electron microscopy of tRNA crystals. II. 4 A resolution diffraction pattern and substantial stability to radiation damage

Electron microscopy was applied to thin crystals of yeast tRNAPhe. The crystals embedded in glucose yield Bragg reflections with a spacing smaller than 4 A. The measurement of radiation damage rate demonstrates that they are 4 to 14 times less susceptible to electron exposures than protein crystals embedded in glucose.     

Scanning electron microscopic and X-ray diffraction studies of otoconia in the lizard Podarcis s. sicula

Summary The otoliths of embryos and young animals of the lizard Podarcis s. sicula were studied by X-ray diffraction and scanning electron microscopy. Two types of crystal that give different X-ray diffraction patterns were found in the membranous labyrinth of Podarcis. The crystals consist of calcite or aragonite and are easily distinguished by scanning electron microscopy because of their different morphology. The two calcium carbonate crystal forms are not mixed at random but are present in the embryo from the very beginning in specific sites. The endolymphatic sac contains aragonite crystals while the saccule contains calcite crystals adjacent to the wall, in addition to a preponderance of aragonite crystals. The utricle and lagena contain only calcite crystals. The presence of two crystal forms of calcium carbonate in the membranous labyrinth are discussed in terms of differing genetic and functional significance.     

Single crystals of V amylose

Single crystals of V amylose were prepared from dilute solution in water/ethanol over a range of temperatures. The effects of crystallization temperature on crystal morphology and thickness were investigated using transmission electron microscopy. Annealing of these crystals gave rise to large increases in crystal thickness. It is concluded that V amylose crystals behave in a similar way to crystals of linear synthetic polymers.     

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.