The iterative helical real space reconstruction method: surmounting the problems posed by real polymers

Many important biological macromolecules exist as helical polymers. Examples are actin, tubulin, myosin, RecA, Rad51, flagellin, pili, and filamentous bacteriophage. The first application of three-dimensional reconstruction from electron microscopic images was to a helical polymer, and a number of laboratories today are using helical tubes of integral membrane proteins for solving the structure of these proteins in the electron microscope at near atomic resolution. We have developed a method to analyze and reconstruct electron microscopic images of macromolecular helical polymers, the iterative helical real space reconstruction (IHRSR) algorithm. We can show that when there is disorder or heterogeneity, when the specimens diffract weakly, or when Bessel functions overlap, we can do far better with our method than can be done using traditional Fourier-Bessel approaches. In many cases, structures that were not even amenable to analysis can be solved at fairly high resolution using our method. The problems inherent in the traditional approach are discussed, and examples are presented illustrating how the IHRSR approach surmounts these problems.     

Three-dimensional reconstruction of the 14-filament fibers of hemoglobin S.

The supramolocular structure of hemoglobin S has been studied by electron microscopy and computer-based image reconstruction. Negatively stained fibers prepared by the lysis of sickled cells or the stirring of hemoglobin S hemolysates have been observed to be almost exclusively of the 20-nm diameter form. These fibers have a periodic variation in diameter between the extremes of 18 nm and 23 nm. Computed Fourier transforms of the fibers show a, highly complex pattern of reciprocal space maxima, with 30 maxima on 20 layer-lines clearly resolved. The Bessel orders of the maxima were assigned with the aid of a newly developed technique, a combined real-space Fourier-space reconstruction method (REFORM). This method utilizes the filtered image produced by the inverse Fourier transform of the low-resolution maxima to calculate in real space the crosssection of a helical fiber. The REFORM analysis indicated that the fibers have an elliptical cross-section and are composed of 14 hexagonally packed filaments with 10 outer filaments surrounding four inner filaments. On the basis of this cross-section, the Bessel orders of all the maxima were assigned, permitting the calculation of three-dimensional reconstructions by Fourier Bessel synthesis. From these reconstructions details of the location of hemoglobin S molecules of each filament were obtained. Hemoglobin S molecules are staggered in adjacent filaments to produce a closely packed helical structure. Reconstructions of fibers at various stages of disassembly revealed a stable intermediate containing 10 filaments which could be characterized in terms of the loss of two pairs of specific outer filaments. A partially disassembled fiber with only six filaments at positions corresponding to three inner and three outer filaments of the parent structure was also identified. The six-filament structure appears to be produced from the 10-filament structure by the loss of two specific pairs of filaments. Thus pairs of filaments are evidently significant structural units in the stabilization of the complete fibers and the orientation of the molecules in these pairs may be related to the filament pairs known to occur in crystals of hemoglobin S.     

Application of the iterative helical real-space reconstruction method to large membranous tubular crystals of P-type ATPases

Since the development of three-dimensional helical reconstruction methods in the 1960's, advances in Fourier-Bessel methods have facilitated structure determination to near-atomic resolution. A recently developed iterative helical real-space reconstruction (IHRSR) method provides an alternative that uses single-particle analysis in conjunction with the imposition of helical symmetry. In this work, we have adapted the IHRSR algorithm to work with frozen-hydrated tubular crystals of P-type ATPases. In particular, we have implemented layer-line filtering to improve the signal-to-noise ratio, Wiener-filtering to compensate for the contrast transfer function, solvent flattening to improve reference reconstructions, out-of-plane tilt compensation to deal with flexibility in three dimensions, systematic calculation of Fourier shell correlations to track the progress of the refinement, and tools to control parameters as the refinement progresses. We have tested this procedure on datasets from Na(+)/K(+)-ATPase, rabbit skeletal Ca(2+)-ATPase and scallop Ca(2+)-ATPase in order to evaluate the potential for sub-nanometer resolution as well as the robustness in the presence of disorder. We found that Fourier-Bessel methods perform better for well-ordered samples of skeletal Ca(2+)-ATPase and Na(+)/K(+)-ATPase, although improvements to IHRSR are discussed that should reduce this disparity. On the other hand, IHRSR was very effective for scallop Ca(2+)-ATPase, which was too disordered to analyze by Fourier-Bessel methods.     

Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy.

Both surfaces of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans were imaged in buffer solution by atomic force microscopy. When adsorbed to freshly cleaved mica, the hydrophilic outer surface of the HPI layer was attached to the substrate and the hydrophobic inner surface was exposed to the stylus. The height of a single HPI layer was 7.0 nm, while overlapping edges of adjacent single layers adsorbed to mica had a height of 14.7 nm. However, double-layered stacks with inner surfaces facing each other exhibited a height of 17.4 nm. These stacks exposed the outer surface to the stylus. The different heights of overlapping layers and stacks are attributed to differences in the interaction between inner and outer surfaces. At high resolution, the inner surface revealed a protruding core with a central pore connected by six emanating arms. The pores exhibited two conformations, one with and the other without a central plug. Individual pores were observed to switch from one state to the other.     

The cell envelope of the hyperthermophilic archaebacterium Pyrobaculum organotrophum consists of two regularly arrayed protein layers: three-dimensional structure of the outer layer

The cell envelope of the hyperthermophilic sulphur-reducing archaebacterium Pyrobaculum organotrophum H10 was found to be composed of two distinct hexagonally arranged crystalline protein arrays. Electron microscopic analysis of freeze-etched cells and isolated envelopes in conjunction with image processing showed that the inner layer (lattice centre-to-centre spacing 27.9 nm) is essentially identical to the protein array of Pyrobaculum islandicum GEO3, a complex, rigid structure implicated in the maintenance of cell shape. The outer layer has clear p6 symmetry and a lattice spacing of 20.6 nm. Its three-dimensional structure was reconstructed from a negative stain tilt series of an intact double-layered envelope using Fourier filtration to separate the desired information from the other lattices present. The outer layer is a unique, porous network of blocklike dimers disposed around six-fold axes, and exhibits minimal asymmetry between its inner and outer faces. It appears to be rather loosely associated with the outer surface of the inner layer. In most H10 envelopes, the inner layer is orientated with one base vector exactly perpendicular to the long axis of the cell, so that the cylindrical portion is composed of a series of parallel cell-girdling hoops of hexameric morphological units. All the other known Pyrobaculum strains were found to have a GEO3-type envelope structure, consisting of a single rigid protein array and a fibrous capsule. Although H10 does not possess a capsule, fibrils appear to be sandwiched between the two protein layers.     

Double-Layered Matrix of Shellac Wax-Lutrol in Controlled Dual Drug Release

Double-layered matrix tablets prepared from shellac wax-lutrol were fabricated using a molding technique, and the release of hydrochlorothiazide and propranolol HCl from the inner tablet or outer layer was studied. The simultaneous determination of dual drug release was measured with first derivative UV spectrophotometry. The tablet containing shellac wax as the outer tablet and lutrol as the inner tablet showed more appropriate drug release and the size of the inner layer influenced the rate of drug release. In addition, the aqueous solubility of the drug and the components of the inner tablet or outer layer affected the drug release behavior. Most of the double-layered tablets exhibited the drug-release pattern which fitted well with zero-order kinetic due to the restriction of the release surface. Biphasic drug release pattern was found in the tablet of which the outer layer rapidly eroded. The drug dissolution data from drug-loaded-outer layer could predict the dissolution time for the outer layer of drug-loaded inner part of double-layered matrix tablet. Incorporation of lutrol increased the drug release from shellac wax matrix, and the zero-order release was attained by fabricating it into a double-layered tablet.     

Tube structure and original composition of Sinotubulites: shelly fossils from the late Neoproterozoic in southern Shaanxi, China

Abundant well-preserved shelly fossils of Sinotubulites occur in the upper part of the late Neoproterozoic Dengying Formation in Ningqiang County, Shaanxi Province, China. Unlike 'funnel-in-funnel' structures of co-occurring Cloudina shells, the tube of Sinotubulites is nearly cylindrical, composed of several thin layers and characterized by 'tube-in-tube' structure. The tube bears prominent longitudinal sculptures and/or irregular annulations, which were formed by the wrinkles of tube layers, and weaken gradually towards the inner layers. The irregular wrinkle ornament of the outer layer shows plastic feature of the Sinotubulites tubes, which may further imply that the organisms secrete the elastic and flexible organic-dominated walls with mineralization, possibly aragonitic. Detailed thin section and SEM studies indicate that the silicification occurred earlier than phosphatation. Specimens preserved in situ from Hubei and surface ornamentation and polygonal shape of the cross-section suggest that Sinotubulites probably lived as an epibenthos lying on the sea floor.     Aragonitic tube, Ediacaran, Shaanxi. China, Sinotubulites , tube structure.     

Superficial Macromolecular Arrays on the Cell Wall of Spirillum putridiconchylium

Electron microscopy of the cell envelope of Spirillum putridiconchylium, using negatively stained, thin-sectioned, and replicated freeze-etched preparations, showed two superficial wall layers forming a complex macromolecular pattern on the external surface. The outer structured layer was a linear array of particles overlying an inner tetragonal array of larger subunits. They were associated in a very regular fashion, and the complex was bonded to the outer, pitted surface of the lipopolysaccharide tripartite layer of the cell wall. The relationship of the components of the two structured layers was resolved with the aid of optical diffraction, combined with image filtering and reconstruction and linear and rotary integration techniques. The outer structural layer consisted of spherical 1.5-nm units set in double lines determined by the size and arrangement of 6- by 3-nm inner structural layer subunits, which bore one outer structural layer unit on each outer corner. The total effect of this arrangement was a double-ridged linear structure that was evident in surface replicas and negatively stained fragments of the whole wall. The packing of these units was not square but skewed by 2 degrees off the perpendicular so that the "unit array" described by optical diffraction and linear integration appeared to be a deformed tetragon. The verity of the model was checked by using a photographically reduced image to produce an optical diffraction pattern for comparison with that of the actual layers. The correspondence was nearly perfect.     

Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans

The low-resolution structure of the regular surface layer of Deinococcus radiodurans has been determined from negatively stained specimens by three-dimensional electron microscopy. The layer has P6 symmetry, a lattice constant of 18 nm and a thickness of 6.5 nm. Three-dimensional reconstruction was performed by a hybrid real space/Fourier space approach that incorporates partial compensation of lattice distortions: The model obtained is discussed in the light of independent information about the surface structure of this layer, derived from metal shadowing and surface relief reconstruction. While agreement is quite satisfactory for the apparently more rigid inner surface, the outer surface shows severe flattening effects. The structure of the HPI layer is compared with other bacterial surface layers using a classification scheme that is outlined in the Appendix.     

Structural polymorphism in bacterial EspA filaments revealed by cryo-EM and an improved approach to helical reconstruction

The traditional Fourier-Bessel approach to three-dimensional reconstruction from electron microscopic (EM) images of helical polymers involves averaging over filaments, assuming a homogeneous structure and symmetry. We have used a real-space reconstruction approach to study the EspA filaments formed by enteropathogenic E. coli. In negative stain, the symmetry of these filaments is ambiguous, and we suggest that such ambiguities may be more prevalent than realized. Using cryo-EM of frozen-hydrated filaments, we find that these filaments have a fixed twist with 5.6 subunits per turn but an axial rise per subunit that varies from about 3.6 A to 5.6 A. Reconstructions at approximately 15 A resolution show a switching between the more compressed and extended filaments in the packing of putative alpha helices around the hollow lumen. Outside of a crystal, where there is nothing to maintain long-range order, the structural polymorphism in helical polymers may be much greater than has been assumed.     

Anatomical Study on the Pedicels of Two Ferns

Pedicel is a stalk connecting sporangia with frond in ferns, and its structure and function are not clear. In this paper, we studied the pedicels of Dryopteris zhuweimingii and Pentarhizidium orientale. We found that the pedicels consist of three lines of cells, and the cell wall can be separated into two layers. The inner layer is secondary cell wall (S1) that spiral and cling to the outer layer. The outer layer is primary cell wall. And when we broke the pedicel into two sections, the inner layer of the cell wall could be pulled out like a spiral belt. This structure may be important to support and protect the sporangia.     

白额鹱卵壳的扫描电镜观察

本文报道白额鹱卵壳的壳膜、孔锥层、海绵层、表层等的超微结构,并对卵壳元素进行ＴＮ－５５００能谱分析。     

Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes

Bacterial cellulose (BC) was deposited in tubular form by fermenting Acetobacter xylinum on top of silicone tubes as an oxygenated support and by blowing different concentrations of oxygen, that is, 21% (air), 35%, 50%, and 100%. Mechanical properties such as burst pressure and tensile properties were evaluated for all tubes. The burst pressure of the tubes increased with an increase in oxygen ratio and reached a top value of 880 mmHg at 100% oxygen. The Young's modulus was approximately 5 MPa for all tubes, irrespective of the oxygen ratio. The elongation to break decreased from 30% to 10-20% when the oxygen ratio was increased. The morphology of the tubes was characterized by Scanning Electron Microscopy (SEM). All tubes had an even inner side and a more porous outer side. The cross section indicated that the tubes are composed of layers and that the amount of layers and the yield of cellulose increased with an increase in oxygen ratio. We propose that an internal vessel wall with high density is required for the tube to sustain a certain pressure. An increase in wall thickness by an increase in oxygen ratio might explain the increasing burst pressure with increasing oxygen ratio. The fermentation method used renders it possible to produce branched tubes, tubes with unlimited length and inner diameters. Endothelial cells (ECs) were grown onto the lumen of the tubes. The cells formed a confluent layer after 7 days. The tubes potential as a vascular graft is currently under investigation in a large animal model at the Centre of Vascular Engineering, Sahlgrenska University     

Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope.     

Novel ultrastructures of Treponema primitia and their implications for motility

Members of the bacterial phylum Spirochaetes are generally helical cells propelled by periplasmic flagella. The spirochete Treponema primitia is interesting because of its mutualistic role in the termite gut, where it is believed to cooperate with protozoa that break down cellulose and produce H₂ as a by-product. Here we report the ultrastructure of T. primitia as obtained by electron cryotomography of intact, frozen-hydrated cells. Several previously unrecognized external structures were revealed, including bowl-like objects decorating the outer membrane, arcades of hook-shaped proteins winding along the exterior and tufts of fibrils extending from the cell tips. Inside the periplasm, cone-like structures were found at each pole. Instead of the single peptidoglycan layer typical of other Gram-negative bacteria, two distinct periplasmic layers were observed. These layers formed a central open space that contained two flagella situated adjacent to each other. In some areas, the inner membrane formed flattened invaginations that protruded into the cytoplasm. High-speed light microscopic images of swimming T. primitia cells showed that cell bodies remained rigid and moved in a helical rather than planar motion. Together, these findings support the 'rolling cylinder' model for T. primitia motility that posits rotation of the protoplasmic cylinder within the outer sheath.     

Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum

The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca2+-ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (approximately 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by approximately 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca2+-ATPase.     

Windex: a toolset for indexing helices

We describe here a set of procedures and algorithms that may be used as an aid in determining the indexing rule of a helical specimen. Crystallizing macromolecules into helical arrays has the potential to speed up and simplify the process of three-dimensional reconstruction of the macromolecular structure. The process of helical reconstruction has been largely automated except for the critical first step of indexing the helical diffraction pattern. This is quite often the rate-limiting step in the overall process, particularly in the case of large helical tubes, which have complicated helical diffraction patterns that may vary from tube to tube. We have developed a set of procedures, supported by a graphical user interface, that provide a straightforward and semi-automated approach to indexing a helical structure. The new procedures have been tested using a number of helical specimens, including TMV, acto-myosin, decorated microtubules, and a variety of helical tubes of a bacterial membrane protein.     

Scanning electron microscope study of cat and dog enamel structure

Scanning electron microscopy revealed several similarities as well as significant differences in the enamel structure between cat and dog teeth. Three enamel layers were present in both species; a surface rodless (aprismatic) layer, an outer layer of parallel rods (only at some sites), and an inner layer with prominent Hunter-Schreger bands. In the inner layer of both carnivores, the diameter of individual rods varied significantly and frequently their course changed abruptly with respect to neighboring rods. In dog teeth the cross-sectional shape of inner enamel rods was pleomorphic, but hexagonal in outer enamel. In contrast, cat enamel rods were rounded in both inner and outer enamel layers. Hunter-Schreger bands of cats circumscribed the teeth in relatively straight segments, but these bands showed pronounced waviness in dog teeth. In cats and dogs the surface rodless layer was structurally continuous with subjacent interrod enamel and covered all tooth surfaces with the exception of the cervical areas. The data show that the structure of inner and outer enamel layers differ between these two carnivore species and that the enamel structure of the cat was most similar to that described in humans. One principal difference between carnivore and human teeth is that the growth lines of carnivores do not terminate at perikymata on the tooth surface.     

A new sampler for extracting undisturbed surface peat cores for growth pot experiments

The sampler extracts uncompressed cores of 13·3 cm in diameter, up to 70 cm long, from the surface layers of peat. It has two close-fitting concentric cylindrical tubes, the outer one acting as a cutter and the inner one as a collector. As the outer tube is introduced by rotation into the peat, the cut core is collected in the inner tube which is maintained in a fixed position during the rotation phase and then pushed down stepwise. This limits friction between the peat core and the wall of the corer and prevents compression or distortion of the peat. These problems are also reduced by means of three skew cutters allowing the peat to be supported during the slicing action. Air can penetrate between the tubes to the lower end of the core, suppressing any suction effect during withdrawal. The sampler has been tested and has worked satisfactorily in many different peat types.