Three-dimensional Crystalline Arrays Research Articles

Analysis of Element Composition of DNA-Protein Crystals In Vitro

The universal response of Escherichia coli to stress is enhancing the synthesis of specific histone-like Dps proteins that bind bacterial DNA. As a result, two-dimensional and three-dimensional crystalline arrays can be observed in the cytoplasm of starving bacteria. Conditions for obtaining in vitro co-crystals of DNA-Dps were selected, and their elemental composition was studied using analytical electron microscopy. It was found that Dps in the co-crystal retains its ferritin-like activity; that is, it can stimulate the oxidation of Fe2+ ions to Fe3+ and facilitate the accumulation of iron in the form of Fe2O3 in the inner cavity of the oligomer.

Projection structures reveal the position of the DNA within DNA-Dps Co-crystals

One of the universal mechanisms for the response of Escherichia coli to stress is the increase of the synthesis of specific histone-like proteins that bind the DNA, Dps. As a result, two-and three-dimensional crystalline arrays may be observed in the cytoplasm of starving cells. Here, we determined the conditions to obtain very thin two-dimensional DNA-Dps co-crystals in vitro, and studied their projection structures, using electron microscopy. Analysis of the projection maps of the free Dps crystals revealed two lattice types: hexagonal and rectangular. We used the fluorescently labeled DNA to prove that the DNA is present within the co-crystals with Dps in vitro, and visualized its position using transmission electron microscopy. Molecular modeling confirmed the DNA position within the crystal. We have also suggested a structural model for the DNA-Dps co-crystal dissolving in the presence of Mg2+ ions.

Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays

Proteins represent the most sophisticated building blocks available to an organism or the laboratory chemist. Yet, in contrast to nearly all other types of molecular building blocks, the designed self-assembly of proteins has been largely inaccessible owing to the chemical and structural heterogeneity of protein surfaces. To circumvent the challenge of programming extensive non-covalent interactions for controlling protein self-assembly, we had previously exploited the directionality and strength of metal coordination interactions to guide the formation of closed, homoligomeric protein assemblies. Here, we extend this strategy to the generation of periodic protein arrays. We show that a monomeric protein with properly oriented coordination motifs on its surface can arrange upon metal binding into one-dimensional nanotubes, and two-or three-dimensional crystalline arrays whose dimensions collectively span nearly the entire nano- and micrometer length scale. The assembly of these arrays is predictably tuned by external stimuli, such as metal concentration and pH.

Thermoresponsive Colloidal Crystals Built from Core−Shell Poly(styrene/α-tert-butoxy-ω-vinylbenzylpolyglycidol) Microspheres

Core-shell particles of poly(styrene/alpha-tert-butoxy-omega-vinylbenzylpolyglycidol) P(S/PGL) were used as new building blocks for the assembly of a colloidal crystal. The added-value properties of these particles for photonic crystal architectures are their high hydrophilicity together with their thermoresponsivity. Indeed, the poglycidol-rich shell undergoes a phase transition above 45 degrees C, which leads to its collapse at the particle surface accompanied by a decrease in the particle diameter. The three-dimensional crystalline arrays display Bragg diffraction properties, as judged by angle-resolved reflectance spectroscopy. The thermoresponsivity of the colloidal assemblies was observed through modifications of their optical properties with respect to the temperature used during the assembly process. The wetting properties of the crystalline material were also shown to reversibly switch from hydrophilic to hydrophobic as a function of the assembly temperature, thus evidencing the reorganization of the surface polyglycidol chains during the polymer phase transition. This work shows conclusively that P(S/PGL) particles are promising alternatives to poly(N-isopropylacrylamide) and poly(ethylene glycol) particles for the elaboration of thermoresponsive colloidal crystals, with a phase transition situated in between those of these two polymers.

Molecular packing of type I collagen in tendon

X-ray diffraction of rat tail tendon shows that type I collagen fibrils contain regions of three-dimensional crystalline arrays; where molecular packing is speculated to be by a staggered sheet or microfibril arrangement. The X-ray diffraction pattern also contains a significant amount of diffuse scatter indicative of static and thermal disorder in fibrils. Removal of the diffuse scatter from the equatorial region of X-ray diffraction patterns obtained using synchrotron radiation allowed the Bragg intensities to be viewed on a flat background. Indexing of Bragg peak intensity on the 10, −10, 0 –1, 01, −11 and 1 –1 row-lines of the triclinic unit cell have been used here to test possible sheet and microfibril packing arrangements. The relative translation of molecular segments in the gap and overlap regions as well as the telopeptide orientation have been investigated. A global search through combinations of molecular packing and molecular translation revealed that the sheet-type conformations cannot account for the observed low-angle off-meridional Bragg peak intensity distribution. A superior fit is obtained with D-staggered left-handed microfibril structures. The orientation of the telopeptides may indicate that there are interconnections between microfibrils that may explain the difficulty in isolating individual microfibrillar structures.

Special Specimen Preparation Methods For Image Processing in Transmission Electron Microscopy: A Review*

One of the important developments in quantitative electron microscopy has been the application of optical and computer imaging methods to electron micrographs. In general these techniques of image analysis have been applied to electron micrographs from isolated biological structures prepared in the presence of various negative stains. To make full use of image processing techniques there are obvious advantages in preparing suitable specimens containing large areas of repeating features. However, the number of naturally occurring biological specimens exhibiting crystalline or paracrystalline features suitable for high resolution electron microscopy and subsequent image analysis is relatively small.Some recent experiments on the in vitro formation of crystalline and paracrystalline arrays from highly concentrated and purified isometric, filamentous and rod-like viruses is reviewed. The problems associated with the preparative procedures for producing two-dimensional and three-dimensional crystalline arrays are discussed together with the possibility of extending the negative staining-carbon film method for studying the gradual dissociation or assembly of viral components.

The in vitro crystalline formations of turnip rosette virus I. Electron microscopy of two- and three-dimensional arrays

Two-dimensional and three-dimensional crystalline arrays of turnip rosette virus (TRosV) have been formed using the negative-staining carbon-film technique for electron microscopy. Three types of packing arrangements of the virus within the lattice arrays have been observed, hexagonal, square, and skewed. Analysis of the electron micrographs, by optical diffraction, showed considerable differences in the packing of the particles compared with other viruses prepared by the same technique. It was found that there was a predominant close hexagonal packing over a wide range of pH values for the first negative stain in which the crystalline arrays were formed on different surface materials. The individual particles seen in the arrays were observed to have a central area of varying contrast. Flotation of the crystalline arrays at the liquid/air surface of a second negative stain at high pH resulted in the gradual disruption of the individual capsids within the lattice.

A negative staining-carbon film technique for studying viruses in the electron microscope: III. The formation of two-dimensional and three-dimensional crystalline arrays of cowpea chlorotic mottle virus

The negative staining-carbon film technique was applied to purified and highly concentrated suspensions of cowpea chlorotic mottle virus (CCMV) wild type and a salt-stable mutant. Both two-dimensional and three-dimensional crystals were rapidly formed at the surface of mica using CCMV suspensions containing about 2.0 to 14.0 mg/ml of virus. When mixed with 3% ammonium molybdate at pH 5.2 the crystals contained CCMV particles predominantly packed in a hexagonal array at the mica surface. At pH 7.0, the crystals were observed to be predominantly in square arrays. Under the preparative conditions used it was possible to form crystalline sheets with sides of several micrometers and in some instances covering entire grid squares. The crystals were found to be ideal for producing low-angle electron diffraction patterns and optical diffraction patterns from the micrographs with spectra extending out to about 2.0 nm. Experiments carried out on CCMV wild type and the salt stable mutants also showed that rapid morphological degradation occurs following storage at 4°C. The viral components from the degraded virus were observed to influence both the formation of the crystals and resolution obtainable from the specimens.

A negative staining-carbon film technique for studying viruses in the electron microscope: II. Application to adenovirus type 5

Two-dimensional and three-dimensional crystalline arrays of adenovirus type 5 were rapidly formed in vitro in the presence of negative stains when applied according to the negative staining-carbon procedure. The adenovirus capsids within the crystal lattice were packed predominantly in hexagonal arrays. Capsids were observed to be packed in positions viewed along axes of threefold symmetry and in other lattice arrays showed capsids in twofold symmetry positions. These crystalline arrays were extended over areas of several hundred microns across and provided ideal specimens for both optical diffraction and low-angle electron diffraction studies. When the adenovirus preparations were subjected to different negative staining and pH conditions, the capsids were observed to disrupt gradually at the specimen support surface. The released components were studied at high resolution and the structural components associated with the hexon and penton capsomeres were resolved. A detailed scale model of the adenovirus capsid was constructed from the information contained in the electron micrographs and the model used to illustrate the possible packing arrangements of the icosahedral capsid and penton fibres in the observed crystalline lattice.

A negative staining—carbon film technique for studying viruses in the electron microscope: I. Preparative procedures for examining icasahedral and filamentous viruses

A simple technique has been developed for the preparation of highly concentrated or crystalline suspensions of viruses in the presence of negative stains. Several icosahedral and filamentous viruses have been studied in concentrations from 1.0–20 mg/ml. The technique has several advantages over conventional negative staining procedures by forming two-dimensional and three-dimensional crystalline arrays of highly concentrated viruses. The packing arrangements of the virus in the crystalline arrays can be varied according to the type of negative stain and pH used during the preparation. High resolution electron micrographs of material prepared by the method have been analysed by optical diffraction procedures. The relative thickness of the carbon supporting film in relation to the physical dimensions of viruses is considerably improved.

Application of the negative staining-carbon technique to the study of virus particles and their components by electron microscopy

The negative staining-carbon technique has been applied to a range of regular (icosahedral) and filamentous (helical) viruses. Under the preparative conditions used, it was demonstrated that the icosahedral particles formed large sheets of two-dimensional or three-dimensional crystalline arrays. Depending on the type of virus, physiological conditions and pH of the virus suspension in the presence of the negative stain solutions, the icosahedral particles could be packed into squares or hexagons. Rod-shaped or filamentous viruses at high concentrations were formed into aggregates extending over large areas with the particles arrange approximately in parallel array. Analysis of high resolution electron micrographs by optical diffraction showed that structural detail present in the images extended down to about 1.8 to 2.0nm. Evidence was obtained indicating the presence of protein and other material in monolayers at the specimen surface which restricted the amount of structural information which could be obtained.

Ultrastructural pathology of coxsackie A4 virus infection of mouse striated muscle

Electron microscopic examination of Coxsackie A4 virus-infected hind limb muscles of suckling mice showed generalized myositis with almost total loss of normal muscle cell architecture. Degenerative changes included dilatation of sarcoplasmic reticulum with concurrent disruption of myofibrils, presenting the appearance of dense amorphous masses in some areas; large accumulations of membrane-bound vesicles; swelling, distortion, and mineralization of mitochondria; increase in lipid deposits; pyknosis of the nuclei; edema and inflammatory infiltration. Virus particles were present in a number of different configurations in muscle fibers: individual particles or small accumulations within vacuoles, two-dimensional linear arrays between membranes, and three-dimensional crystalline arrays, usually in close apposition to sarcoplasmic reticulum or sarcolemma.