Flexibility observed in high resolution structures of <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> ribonucleases determined by diffraction methods

It has been widely accepted to distinguish between static structures determined by diffraction methods and dynamic structures determined by nuclear magnetic resonance (NMR). The dynamics of NMR structures is demonstrated by an ensemble of a number of overlaid structures. This cannot be seen in one structure determined by diffraction methods. However, it is possible to see the flexibility of a protein molecule in a number of structures of the same protein determined by X-ray techniques which is manifested by different conformations of main-chain. Multiple protein structure determination does not provide identical structures as a result of various factors including flexibility. Overlap of structures of a protein determined at atomic resolution with high accuracy shows that the root-mean-square deviations (rmsd) of main-chain atoms exceed several fold the accuracy of the positional parameters of each structure. Overlap of a number of structures of a protein determined by diffraction methods shows a similar distribution as that determined by NMR. These observations are demonstrated using high resolution structures of Streptomyces aureofaciens ribonucleases, their mutants and complexes with ligands.     

Enlarged representative set of protein structures.

To reduce redundancy in the Protein Data Bank of 3D protein structures, which is caused by many homologous proteins in the data bank, we have selected a representative set of structures. The selection algorithm was designed to (1) select as many nonhomologous structures as possible, and (2) to select structures of good quality. The representative set may reduce time and effort in statistical analyses.     

Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.     

Tubular structures occurring in cells infected with herpesviruses

Certain viruses belonging to the Herpesviridae family generate tubular structures of three distinct size ranges late in the growth cycle. The smallest tubules are about 10 to 20 nm in diameter, and are restricted to the cell nucleus, tending to form palisades of lattice-like structures. The medium sized tubular structures are about the width of a core particle, measuring 55 to 65 nm in diameter, while the larger tubular structures are the same diameter as viral capsids, about 80 to 100 nm. Both are rigid capsomered structures, which are sometimes encountered outside the cell nucleus budding onto spherical particles. The available data on some properties of the various tubular structures, as well as speculations concerning their nature and significance are briefly reviewed. Their importance is emphasized for antigen and vaccine production, as well as in differentiating herpesviruses by electron microscopy.     

A comparison of the restrained molecular dynamics and distance geometry methods for determining three-dimensional structures of proteins on the basis of interproton distances

A direct comparison of the metric matrix distance geometry and restrained molecular dynamics methods for determining three-dimensional structures of proteins on the basis of interproton distances is presented using crambin as a model system. It is shown that both methods reproduce the overall features of the secondary and tertiary structure (shape and polypeptide fold). The region of conformational space sampled by the converged structures generated by the two methods is similar in size, and in both cases the converged structures are distributed about mean structures which are closer to the X-ray structure than any of the individual structures. The restrained molecular dynamics structures are superior to those obtained from distance geometry as regards local backbone conformation, side chain positions and non-bonding energies.     

Atomic resolution crystal structures of HIV-1 protease and mutants V82A and I84V with saquinavir

Due to the limited distance data available from the experiments, the structures determined by NMR Spectroscopy may not always be as accurate as desired. Further refinement of the structures is often required and sometimes critical. With the increase of high quality protein structures determined and deposited in PDB Data Bank, commonly shared protein conformational properties can be extracted based on the statistical distributions of the properties in the structural database and used to improve the outcomes of the NMR-determined structures. Here we examine the distributions of protein interatomic distances in known protein structures. We show that based on these distributions, a set of mean-force potentials can be defined for proteins and employed to refine the NMR-determined structures. We report the test results on 70 NMR-determined structures and compare the potential energy, the Ramachandran plot, and the ensemble RMSD of the structures refined with and without using the derived mean-force potentials.     

Microbially induced sedimentary structures indicating climatological, Hydrological and depositional conditions within recent and pleistocene coastal facies zones (Southern Tunisia)

Summary Extensive tidal areas of the Recent coast of southern Tunisia are overgrown by microbial mats. Different mat types of which each are dominated by distinct and well adapted cyanobacterial species develop. Ecological response of the mat-forming microorganisms to climatological hydrological and sedimentological factors produce characteristic sedimentary structures (=microbially induced sedimentary structures). A suecession of Pleistocene rocks crops out near the lagoon El Bibane, southern Tunisia. The stratigraphic section comprises structures that we regard as fossil equivalents to those microbially induced structures we observe in the Recent coastal area. Preservation of the structures is result of lithification of the microbial mats. This we conclude from fossil filaments of cyanobacteria visible within the rock matrix. The Recent microbially induced sedimentary structures indicate facies zones within the modern tidal environment. Comparison of the Recent structures with the fossil analogues recorded in the stratigraphic section aids to identify the same distinct facies zones within the Pleistocene coastal environment also. Erosion by water currents forms step-like cliffs, and the microbial mat is undermined and ripped off piece by piece. shallows within the supratidal area are overgrown by copious microbial mats comprising structures like biolaminites and—varvites, as well as polygons of cracks. The features originate from effects triggered by seasonal variations of climate. Tufts and reticulate pattern of bulges indicate supernatant water films covering the mat surfaces. Morphologically higher parts of the Recent tidal area are overgrown by single-layered mats forming petees, induced by microbial mat growth and evaporitive pumping. The study demonstrates that microbially induced sedimentary structures can be used to reconstruct small-scaled facies zones within coastal environments. The also include hints on paleoclimatological, hydrological and sedimentological conditions.     

Dynamics of the formation of secondary reproductive structures in Streptomyces zone-forming colonies as determined from confocal microscopy data

The dynamics of the formation of secondary structures on the colony surface, so-called air mycelium concentric rings, was investigated by confocal microscopy and using Streptomyces zone-forming colonies as a model of a self-organizing system. The occurrence of luminescence in reproductive structures was registered, whereas in vegetative structures it was almost completely absent. It was shown that secondary reproductive structures begin to form within the thickness of substratum mycelium as branches or as an apical extension of vegetative gyphes, followed by the formation of numerous secondary branches filled with protospores.     

The difference between protein structures that are obtained by X-ray analysis and magnetic resonance spectroscopy

We have analyzed the proteins whose structures were determined both by X-ray analysis (X-ray) and nuclear magnetic resonance (NMR) on condition that these structures do not differ greatly when spatially superimposed on each other (61 pairs of protein structures). Atom-atomic contacts (contact distances varied from 2 to 8 A) have been analyzed and it has been found that NMR structures (in comparison with X-ray ones) have more contacts in the range below 3.5 A and above 5.5 A. In the case of residue-residue contacts NMR structures have more contacts below 3 A and between 4.5 and 6.5 A. At all the other contact distances analyzed the X-ray structures have more contacts. The difference in the number of atom-atomic and residue-residue contacts is greater for internal residues, that are concealed from water, as compared to the surface residues. The other, not less important difference deals with the number of hydrogen bonds in the main chain: it is larger for the X-ray structures. The correlation between the hydrogen bonds identified in the structures obtained by both methods is no more than 32%. The consideration of a complete set of protein structures obtained by NMR results in the fact that the number of hydrogen bonds grows 1.2 times as compared to those obtained with the X-ray analysis, whereas the correlation increases only by 65%. We have also demonstrated that alpha-helices in the NMR structures are more distorted in comparison with the ideal alpha-helix, than alpha-helices in the X-ray structures.     

THE MEMBRANE SYSTEM OF STREPTOMYCES CINNAMONENSIS

The plasma membrane bounding the cytoplasm immediately inside the hyphal wall of Streptomyces cinnamonensis may not retract from the hyphal wall. When it does retract from the wall, it appears as a single dark line in some sections and as 2 dark lines separated by a light zone in others. The membrane system consists of mesosomes and endomembrane structures. The mesosomes are those membrane structures whose derivatives appear to be the plasma membrane. The endomembrane structures, in the present report, are those that appear to have been derived from either the cytoplasm or the limiting membranes of the pre-existing membrane structures. All membranes seem capable of proliferation, a mechanism obviously responsible for the growth of the individual membrane structures and for the origin of many new ones. The mesosomes, according to their limits, are of 2 distinct types, the open mesosomes and the closed mesosomes. The open mesosomes are partially enclosed by limiting membranes, leaving the unenclosed sides limited by the wall. These mesosomes, when old, usually in aerial hyphae, may become attached to the wall and somewhat deleted from their limiting membranes. The individual membranes in their interior may appear disfigured. The closed mesosomes are completely surrounded by the limiting membranes. These mesosomes, as well as endomembrane structures, retain their original positions in the cytoplasm even in the older aerial hyphae, and the membranes in their interior usually remain practically as distinct in the aerial hyphae as they are in the substratal hyphae. New mesosomes and endomembrane structures are being formed continuously as the mycelium develops. The mesosomes, as a rule, occupy more or less the peripheral regions of the cytoplasm, while the endomembrane structures distribute themselves widely in the cytoplasm and also in the nucleoids. The appearance of the unit membranes, being double-layered (2 dark lines separated by a light line), is not consistent. The membranes as a whole are more resistant to degeneration than the cytoplasm and the nucleoids.     

Metabolic characteristics of brain structures during conscious and unconscious anxiety

Metabolic processes accompanying conscious and unconscious anxiety states in patients with multiple sclerosis were studied. The correspondence between glucose metabolism rates (evaluated using positron emission tomography images) and indices of conscious and unconscious anxiety (according to the Taylor and Luscher test scores) was estimated. Data on the specific features of brain activity at various levels of conscious and unconscious anxiety were obtained. The elevation of conscious anxiety was accompanied by an increase in the metabolic activity of the left-hemisphere cortical and subcortical structures, as well as by a decrease in the glucose metabolism rate in the majority of right-hemisphere structures (except limbic and thalamic structures). The elevation of unconscious anxiety was accompanied by a decrease in the rate of glucose metabolism in the left frontal structures, whereas in the amygdala, as well as in the other limbic structures (gyrus temporalis inferior), its rate increased.     

Unusual lysosomes in aortic smooth muscle cells: presence in living and rapidly frozen cells

Unusual tubular structures have been observed in rat aortic smooth muscle cells (SMC) grown in culture. These tubular structures have several characteristics that strongly suggest that they are lysosomes: they are bounded by a single membrane bilayer, contain densely staining material, and acid phosphatase activity. Furthermore, these structures are present in living cells, as demonstrated by their ability to accumulate the membrane-impermeable fluorescent dye lucifer yellow CH. In ultrastructural preparations they are best seen in samples that are cryofixed by rapid freezing and then freeze-substituted in osmium-acetone solutions. Conventional chemical fixation did not appear to preserve these structures to as great an extent as did rapid freezing. Comparison of SMC in vitro to the same cells in situ revealed differences in lysosome number as well as morphological appearance. Thus, the culturing of rat SMC leads to the formation of unusual tubular lysosomes whose ultrastructural appearance is particularly sensitive to the methods employed for examination.     

Protein docking using surface matching and supervised machine learning

Computational prediction of protein complex structures through docking offers a means to gain a mechanistic understanding of protein interactions that mediate biological processes. This is particularly important as the number of experimentally determined structures of isolated proteins exceeds the number of structures of complexes. A comprehensive docking procedure is described in which efficient sampling of conformations is achieved by matching surface normal vectors, fast filtering for shape complementarity, clustering by RMSD, and scoring the docked conformations using a supervised machine learning approach. Contacting residue pair frequencies, residue propensities, evolutionary conservation, and shape complementarity score for each docking conformation are used as input data to a Random Forest classifier. The performance of the Random Forest approach for selecting correctly docked conformations was assessed by cross-validation using a nonredundant benchmark set of X-ray structures for 93 heterodimer and 733 homodimer complexes. The single highest rank docking solution was the correct (near-native) structure for slightly more than one third of the complexes. Furthermore, the fraction of highly ranked correct structures was significantly higher than the overall fraction of correct structures, for almost all complexes. A detailed analysis of the difficult to predict complexes revealed that the majority of the homodimer cases were explained by incorrect oligomeric state annotation. Evolutionary conservation and shape complementarity score as well as both underrepresented and overrepresented residue types and residue pairs were found to make the largest contributions to the overall prediction accuracy. Finally, the method was also applied to docking unbound subunit structures from a previously published benchmark set.     

Structural organization of kinetoplast DNA and its compaction in the in vitro model system.

Electron microscopic studies of Leishmania gymnodactyli cells lysed at hypotonic conditions showed that the structures identified as kinetoplast DNA have the appearance of loose accumulations of crossed and sometimes branched rod-like structures 100 to 200 nm long and 20 nm thick. The compaction of isolated kinetoplast DNA (kpDNA) caused by interaction with synthetic tripeptide--dansylhydrazide trivaline--was also studied. The analysis of the structures arising at different steps of compaction showed that the minicircles are compacted forming rod-like structures where minicircle double-stranded DNA segments are closely associated side by side in a manner which was earlier described for initial compaction stages of "triple rings". These rod-like structures resemble in their appearance the structures found in lysed cell preparations obtained according to Miller's method. Branching of rod-like structures can be the consequence of minicircle catenation. In vitro compaction is completed with the formation of a compacted network, its diameter being 3 to 6 times smaller as compared with the initial one.     

Computer analysis of potential stem structures of rRNA operons in various procaryote genomes

The processing of 16S rRNA and 23S rRNA by RNase III in E.coli is known to involve stem structures formed by both ends of the rRNA. Indeed, complementary nucleotide sequences are usually found at both ends of 16S rRNA and 23S rRNA. However, whether or not this phenomenon exists in various other bacteria has not yet been adequately studied. We have conducted computer analyses of potential stem structures of rRNA operons in 12 bacterial and 3 archaeal genomes, and compared characteristics of the stem structures among these species. We systematically computed free energy values by exhaustively 'annealing' sequences around the 5' end and sequences around the 3' end of both 16S rRNA and 23S rRNA genes, in order to predict potential stem structures.The results suggest that rRNAs in most species form stem structures at both ends. Some species, such as A.aeolicus, seem to form unusually stable stem structures. On the other hand, some rRNAs, such as rRNAs of D.radiodurans, seem not to form solid stem structures. This suggests that rRNA processing in those species must employ a reliable targeting mechanism other than recognizing stem structures by RNase III.     

NMR studies of bipyrimidine cyclobutane photodimers

Cyclobutane-type photodimers of dinucleoside monophosphates dCpdT, dTpdC and dTpdT were prepared by ultraviolet irradiation in the presence of acetophenone as photosensitizer. The cytosine-containing derivatives were found to deaminate forming uracil products. Using one- and two-dimensional NMR, the photoproducts were characterized as cis-syn and trans-syn cyclobutane photodimers. On the basis of NOE data the structures of the cis-syn and trans-syn products of dUpdT were determined using distance-geometry and restrained-energy-minimization methods. The cis-syn structures showed (high-ANTI/SYN)/high-ANTI glycosidic linkages while the trans-syn structures were in the SYN-ANTI region. The backbone conformations of both structures were in fair agreement with the coupling-constant-data. The trans-syn structures were found to be very rigid and similar in all three products. For the three cis-syn structures more conformational freedom and more variation among the three structures was observed.     

Position of origin of donor posterior chick wing bud tissue transplanted to an anterior host site determines the extra structures formed

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.     

Secondary structure formation during RNA synthesis.

We observed the secondary structures that formed in an RNA molecule during its synthesis. Some of the secondary structures seen in nascent chains were observed to form, then to dissociate in favor of an alternative structure, and then to reform, as chain growth continued. The results show that secondary structures in an RNA molecule are in a state of dynamic equilibrium, and that the extension of a sequence by chain growth, or the reduction of a sequence by processing, may result in significant changes in the secondary structures that are present.