Patch averaging of electron images of GP3*I crystals with variable thickness.

The combination of Fourier and correlation averaging techniques with multivariate statistical analysis and classification, a method known as patch averaging, is used to analyze untilted and tilted images of negatively stained GP32*I crystals, which exhibit variable thicknesses in a single crystal. Within a single image, coherent areas of the same apparent thickness can be distinguished from areas of differing thicknesses. Analysis using the phase relationships among symmetry-related reflections from reconstituted images obtained from untilted micrographs confirms the ability of the method to classify these variable thicknesses properly. Furthermore, the phases from some of the reconstituted images obtained from both untilted and tilted micrographs were found to match well with the phases in a previously determined three-dimensional data set of this crystal with pg symmetry along the crystallographic b axis. These results indicate the utility of the patch averaging procedures in the structural determination of protein crystals with different thicknesses.     

Semiconductor nanocrystal probes for human metaphase chromosomes

To improve signal stability and quantitation, an optically stable, novel class of fluorophore for hybridization analysis of human metaphase chromosomes is demonstrated. Detection of hybridization sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide [(CdSe)ZnS]. Fluorescence of nanocrystal fluorophores was significantly brighter and more photostable than organic fluorophores Texas Red and fluorescein. Thus, semiconductor nanocrystal fluorophores offer a more stable and quantitative mode of fluorescence in situ hybridization (FISH) for research and clinical applications.     

Morphological change of cell-membrane-integrated crystalline structure induced by cell shape change inEuglena gracilis

Summary Fourier transform and an image filtering technique were used for structural analysis of the pellicular strip inEuglena gracilis. Freeze-fracture images of a two-dimensional crystalline structure in the plasma membrane were taken from elongated and rounded cells. Their lattice constants were precisely determined from Fourier transform patterns, and masked filter images were generated. Although differences in their lattice constants could not be detected, the Fourier transform patterns showed a significant difference between these two stages of cell shape. In elongated cells, the pseudo-crystal lattice was generated in the half periodicity of the minor striation. In contrast, it disappeared when the cells became rounded. The filtered images confirmed the crystallographic disagreement between these two stages in cell shape, probably reflecting morphological changes of integrated plasma membrane particles. Four particles detected in a unit structure showed quite similar shapes across the pseudo-lattice in elongated cells, though a unit structure in rounded cells consisted of four heterogeneous particles with opaque boundaries. A 39 kDa integral plasma membrane protein (IP39) is known as a major component in the plasma membrane ofE. gracilis. The compositions of monomeric and oligomeric forms of IP39 were determined in both elongated and rounded cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the possibility that an oligomerization of IP39 proceeds during rounding-up movement of the cell. These morphological and stoichiometrical changes of the integrated plasma membrane proteins may suggest an active involvement in cell shape change.     

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.     

Averaging data derived from images of helical structures with different symmetries.

There are many examples of macromolecules that form helical tubes or crystals, which are useful for structure determination by electron microscopy and image processing. Helical crystals can be thought of as two-dimensional crystals that have been rolled into a cylinder such that two lattice points are superimposed. In many real cases, helical crystals of a particular macromolecule derive from an identical two-dimensional lattice but have different lattice points superimposed, thus producing different helical symmetries which cannot be simply averaged in Fourier-space. When confronted with this situation, one can select images corresponding to one of the observed symmetries at the expense of reducing the number of images that can be used for data collection and averaging, or one can calculate separate density maps from each symmetry, then align and average them together in real-space. Here, we present a third alternative, which is based on averaging of the Fourier-Bessel coefficients, gn,l(r), and which allows the inclusion of data from all symmetry groups derived from a common two-dimensional lattice. The method is straightforward and simple in practice and is shown, through a specific example with real data, to give results comparable to real-space averaging.     

Selenium detoxification of heavy metals: a possible mechanism for the blood plasma

Among the activities of the essential trace element selenium is the ability to reduce the toxicity of heavy metal ions like cadmium(II) and mercury(II). Detoxification often depends on the metabolic reduction of selenium to hydrogen selenide; the mechanism generally advanced to explain such selenium/metal interactions is that selenide combines with heavy metal ions to give a metal selenide which is metabolically inert. However, this hypothesis does not consider circumstances where selenide is quickly removed by other reactions. Given the ease with which selenide is oxidized, such conditions are likely to occur in the blood plasma, an environmental rich in oxidizing agents and a site for many selenium/metal interactions. Using polarography to monitor both selenide and cadmium, we have found that selenide reacts rapidly in vitro with the disulfide bonds present in bovine serum albumin in preference to forming cadmium selenide. We hypothesize that a similar reaction occurs in the blood plasma with the disulfide bonds of plasma proteins to generate thiol groups on the protein involved, and that these newly formed thiols are responsible for the observed reduction of metal toxicity through the ability to chelate heavy metal ions.     

Synthesis and characterization of cadmium selenide quantum dots doped by europium and investigation of their chemiluminescence properties and antibacterial activities

Nanoparticles of cadmium selenide (CdSe) doped with europium, were synthesized as stabilizing agents using thioglycolic acid ligand. This method is based on the enhancing effect of CdSe quantum dots (QDs) doped with europium on chemiluminescence (CL) emission. This emission was generated by mixing CdSe QDs with manganese (II), iron (II) and chrome (II) sulfates as catalysts in the presence of hydrogen peroxide (H₂O₂). The structural characteristics and morphology of these nanoparticles were investigated by scanning electron microscopy, Fourier transform infrared spectroscopy, ultraviolet–visible absorption spectroscopy, X‐ray pattern and dynamic light scattering methods. The CdSe QDs doped with europium were used as the sensitizer in a luminol?hydrogen peroxide CL system. The sensitized CdSe QDs were analyzed for antibacterial activity against Gram‐positive or Gram‐negative bacteria. The results showed that the CdSe QDs are effective against all the studied bacteria, effectiveness was especially higher for Bacillus subtilis.     

Crystal structure and thermal characterization of cadmium oxalate [CdC2O4 · 3H2O] and barium-doped cadmium oxalate [Ba0.5Cd0.5(C2O4)2 · 5H2O] single crystals grown in silica gel

Pure cadmium oxalate trihydrate (COT) and barium added cadmium oxalate (BCO) single crystals were grown by controlled diffusion of Cd²⁺ and Ba²⁺ ions in silica gel at ambient temperature. A single test tube technique coupled with gel aging conferred maximum size crystals by controlling the nucleation rate. It was found that the pH and age of the gel greatly influenced the crystal quality, their size and transparency. Grown crystals CdC₂O₄ · 3H₂O and Ba_0.5Cd_0.5(C₂O₄)₂ · 5H₂O were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and thermal analysis. Effect of barium dopant on the growth and morphology of cadmium oxalate was studied. Pure cadmium oxalate crystallized in triclinic system and the barium-doped cadmium oxalate crystallized in hexagonal system with massive changes in their unit cell parameters. The infrared spectrum revealed the presence of oxalate ligands and water of hydration in both the pure and barium-doped crystals. Thermal analysis showed that the grown crystals were dehydrated thermally even from lower temperatures and the doped crystals were found more stable.     

The relative importance of local phase and local amplitude in patchwise image reconstruction

Natural images were subjected to patchwise Fourier analysis, and the local amplitude and phase spectra were swapped between different images. When the patches were large relative to the image size, the appearance of the reconstructed image was similar to that of the image from which the phase information had been derived, in agreement with previous reports of phase-dominance in the global Fourier Transform. However, when the patch size was made sufficiently small, the appearance of reconstructed images was dominated by amplitude rather than phase. This was not simply due to the DC component of the amplitude spectrum. Prior low-pass filtering of the images enhanced the dominance of amplitude information in the patchwise transform. We conclude that patchwise-reconstructed images contain two quite distinct kinds of information for the human observer. The first is the positional information (local sign) of the patches themselves; the second is the textural information within patches, which is dominated by amplitude rather than phase. The reason why the global Fourier Transform is dominated by phase is that in the absence of any other information about local sign, phase is necessary to reconstruct localised features such as edges.     

Immobilization of cadmium in soil by microbially induced carbonate precipitation with Exiguobacterium undae at low temperature

Microbially induced carbonate precipitation (MICP) is an advanced biological treatment technology to immobilize heavy metals in form of carbonate salts. In this MICP study, ureolytic Exiguobacterium undae was employed for immobilization of cadmium in contaminated soil at low temperature (10 °C). The sequential extraction test revealed conversion of more than 90% of cadmium in the tested soil from the soluble-exchangeable fraction to carbonate-bound fraction in 14 days of treatment. The cadmium may be precipitated in a separate CdCO₃ phase or be co-precipitated in calcite crystals. Activities of urease and dehydrogenase were enhanced during MICP, which were not affected by the testing temperatures. MICP with E. undae is a biological approach that may be worth investigating further to immobilize cadmium in soils of cold regimes.     

Ligand-linked structural transitions in crystals of a cooperative dimeric hemoglobin

Cooperative ligand binding in the dimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis is mediated primarily by tertiary structural changes, but with a small quaternary rearrangement (approximately 3 degrees), based on analysis of distinct crystal forms for ligated and unligated molecules. We report here ligand transition structures in both crystal forms. Binding CO to unligated HbI crystals results in a structure that approaches, but does not attain, the full allosteric transition. In contrast, removing CO from the HbI-CO crystals results in a structure that possesses all the key low affinity attributes previously identified from analysis of HbI crystals grown in the unligated state. Subsequent binding of CO shows the reversibility of this process. The observed structural changes include the quaternary rearrangement even under the constraints of lattice interactions, demonstrating that subunit rotation is an integral component of the ligand-linked structural transition in HbI. Analysis of both crystal forms, along with data from HbI mutants, suggests that the quaternary structural change is linked to the movement of the heme group, supporting a hypothesis that the heme movement is the central event that triggers cooperative ligand binding in this hemoglobin dimer. These results show both the effects of a crystal lattice in limiting quaternary structural transitions and provide the first example of complete allosteric transitions within another crystal lattice.     

Two-dimensional structure of plant light-harvesting complex at 3.7 A [corrected] resolution by electron crystallography

The structure of the light-harvesting chlorophyll a/b-protein complex has been determined at 3.7 A resolution in projection by electron diffraction, electron microscopy and image analysis. Diffraction patterns and high-resolution spotscan images of two-dimensional crystals stabilized with tannin were recorded at low temperature. Phases of structure factors were obtained directly by image processing, after correction of the images for lattice distortions, defocus and beam tilt. Amplitudes were measured by electron diffraction. The projection map shows the detailed structure of the trimeric complex, suggesting the positions of two domains of potential structural and functional homology, of one membrane-spanning alpha-helix approximately perpendicular to the membrane plane and of several tightly bound lipid molecules.     

High resolution electron microscope lattice images of powdered gallstones

Atomic structural images of gallstones were characterized by high-resolution electron microscopy. Regardless of the types of gallstones there was a broad spectrum of crystal size and perfection observed in the stone particles. The crystalline zones in the stone matrix were characterized by well-defined, strongly bounded, ordered arrangement of atoms or atom clusters, while the amorphous zones were characterized by an interlacing network of granulo-fibrillar substructures. The granulo-fibrillar substructures in the amorphous zone could be seen to bond tightly to the lattice fringes in the crystalline zone along the interfaces between two zones. The crystal lattices at the boundary between the crystals were very well matched with the periodicity of atoms continuous through the two crystals. It is hoped that these new electron microscopic data principally related to the structural organization in the gallstones at the atomic or near-atomic level are helpful to a more complete understanding of the micromechanisms of gallstone formation.     

The digital structural analysis of helical hirano bodies obtained by electron microscopy

A digital structural analysis method with the aid of a scanning densitometer was successfully applied to paracrystalline inclusion bodies which have been found in the brains of humans exhibiting senile dementia.Based on Fourier techniques involving noise removal and enhancement for structural analysis from a computer-generated diffractogram, it is proposed that the crystalline bodies from thin sections of the specimen constitute a stranded helix of 18.5 nm pitch.From the digital processed data obtained from the various different electron micrographs obtained by the use of a tilting device, a three-dimensional model and Fourier synthetic images representing the precise arrangement of the Hirano body stranded helix are derived and graphically demonstrated.     

Electron crystallographic analysis of two-dimensional crystals of sensory rhodopsin II: a 6.9 A projection structure

Sensory rhodopsins, phototaxis receptors in Haloarchaea, were purified and reconstituted into halobacterial lipids to form photoactive two-dimensional crystals. Images of vitreous ice-embedded, flattened, tubular crystals of sensory rhodopsin II (SRII) of Natronobacterium pharaonis were recorded using a field emission gun electron cryo-microscope. Fourier components for the SRII structure were determined either from the separated image transforms from single layers that formed each side of flattened tubes, or by a deconvolution procedure when two layers were stacked in register so that they generated a single crystal lattice by superposition. Most micrographs showed significant diffraction to 6.9 A after computer processing, and the results provide the first intermediate- resolution information obtained for an archaeal sensory rhodopsin. The projection structure of SRII indicates that the helix positions match the seven-helix arrangement of the archaeal transport rhodopsins rather than that of the eukaryotic visual pigments. The structural similarity of SRII to the transport rhodopsins supports models in which the transport and signalling mechanisms of archaeal rhodopsins derive from the same retinal-driven changes in protein conformation.     

Single-particle approaches in the analysis of small 2D crystals of the mitochondrial channel VDAC

It has been difficult to obtain better than moderate resolution in analysis of electron microscopic images of small, 2D crystals with variable lattice parameters, e.g., crystals of the channel VDAC generated by phospholipase treatment of outer mitochondrial membranes. We demonstrate that applying single-particle analysis methods to correlation-averaged images can lead to significant improvements in the attainable resolution. Application of a soft-edged fitted mask passing only the central unit cell, and excluding the positionally variable adjacent unit cells, allows improved alignment and more sensitive multivariate statistical analysis, needed to guide intelligent merging of data from different crystals.     

The Use of Electron Tomography for Structural Analysis of Disordered Protein Arrays

A method based on Fourier transforms is described for obtaining a 3-D reconstruction from a paracrystalline object with static disorder. The method is derived from the standard methods used in 3-D reconstruction of 2-D crystals except that all of the Fourier coefficients are used and not just the sampled data from the periodic lattice. Thus, not only is the spatially ordered part of the structure visualized in 3-D, but also the spatially disordered part. Application of the method to 3-D reconstructions of insect flight muscle is described as well as prospects for extension of the method to radiation-sensitive specimens.     

Relationship between bone tissue strain and lattice strain of HAp crystals in bovine cortical bone under tensile loading

Cortical bone is a composite material composed of hydroxyapatite (HAp) and collagen. As HAp is a crystalline structure, an X-ray diffraction method is available to measure the strain of HAp crystals. However, HAp crystals in bone tissue have been known to have the low degree of crystallization. Authors have proposed an X-ray diffraction method to measure the lattice strain of HAp crystals from the diffusive intensity profile due to low crystallinity. The precision of strain measurement was greatly improved by this method. In order to confirm the possibility of estimating the bone tissue strain with measurements of the strain of HAp crystals, this work investigates the relationship between bone tissue strain on a macroscopic scale and the lattice strain of HAp crystals on a microscopic scale. The X-ray diffraction experiments were performed under tensile loading. Strip bone specimens of 40x6x0.8mm in size were cut from the cortical region of a shaft of bovine femur. A stepwise tensile load was applied in the longitudinal direction of the specimen. By detecting the diffracted X-ray beam transmitted through the specimen, the lattice strain was directly measured in the loading direction. As a result, the lattice strain of HAp crystals showed lower value than the bone tissue strain measured by a strain gage. The bone tissue strain was described with the mean lattice strain of the HAp crystals and the elastic modulus.     

Structure of light-harvesting chlorophyll a/b protein complex from plant photosynthetic membranes at 7 A resolution in projection

The structure of thin three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex, an integral membrane protein from the photosynthetic membrane of chloroplasts, has been determined at 7 A (1 A = 0.1 nm) resolution in projection. The structure analysis was carried out by image processing of low-dose electron micrographs, and electron diffraction of thin three-dimensional crystals preserved in tannin. The three-dimensional crystals appeared to be stacks of two-dimensional crystals having p321 symmetry. Results of the image analysis indicated that the crystals were disordered, due to random translational displacement of stacked layers. This was established by a translation search routine that used the low-resolution projection of a single layer as a reference. The reference map was derived from the symmetrized average of two images that showed features consistent with the projected structure of negatively stained two-dimensional crystals. The phase shift resulting from the displacement of each layer was corrected. Phase shifts were then refined by minimizing the phase residual, bringing all layers to the same phase origin. Refined phases from different images were in agreement and reliable to 7 A resolution. A projection map was generated from the averaged phases and electron diffraction amplitudes. The map showed that the complex was a trimer composed of three protein monomers related by 3-fold symmetry. The projected density within the protein monomer suggested membrane-spanning alpha-helices roughly perpendicular to the crystal plane. The density in the centre and on the periphery of the trimeric complex was lower than that of the protein, indicating that this region contained low-density matter, such as lipids and antenna chlorophylls.     

Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I.

Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.