scholarly journals 3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity

2011 ◽  
Vol 108 (51) ◽  
pp. 20544-20549 ◽  
Author(s):  
M. J. Landsberg ◽  
S. A. Jones ◽  
R. Rothnagel ◽  
J. N. Busby ◽  
S. D. G. Marshall ◽  
...  
Keyword(s):  
Insecticidal Activity ◽  
3D Structure ◽  
Toxin Complex

Purification and Characterization of a High-Molecular-Weight Insecticidal Protein Complex Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens

1998 ◽  
Vol 64 (8) ◽  
pp. 3029-3035 ◽  
Author(s):  
David J. Bowen ◽  
Jerald C. Ensign
Keyword(s):  
Molecular Weight ◽  
Insecticidal Activity ◽  
Protein Complex ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Toxin ◽  
The Family ◽  
Toxin Complex

ABSTRACT Photorhabdus luminescens is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family Heterorhabditidae. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases P. luminescens cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of, Manduca sexta larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the classInsecta.

scholarly journals Activity of a Holin-Endolysin System in the Insecticidal Pathogenicity Island ofYersinia enterocolitica

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Katharina Springer ◽  
Sandra Reuter ◽  
Mandy Knüpfer ◽  
Lukas Schmauder ◽  
Philipp-Albert Sänger ◽  
...  
Keyword(s):  
Insecticidal Activity ◽  
High Specificity ◽  
Pathogenicity Island ◽  
Open Reading Frames ◽  
Content Type ◽  
Toxin Complex ◽  
Genes Encoding ◽  
Conserved Genes ◽  
Nematocidal Activity ◽  
Lysis Cassette

ABSTRACTYersinia enterocoliticais a pathogen that causes gastroenteritis in humans. Because of its low-temperature-dependent insecticidal activity, it can oscillate between invertebrates and mammals as host organisms. The insecticidal activity of strain W22703 is associated with a pathogenicity island of 19 kb (Tc-PAIYe), which carries regulators and genes encoding the toxin complex (Tc). The island also harbors four phage-related and highly conserved genes of unknown functions, which are polycistronically transcribed. Two open reading frames showed significant homologies to holins and endolysins and exhibited lytic activity inEscherichia colicells upon overexpression. When a set ofYersiniastrains was tested in an equivalent manner, highly diverse susceptibilities to lysis were observed, and some strains were resistant to lysis. If cell lysis occurred (as demonstrated by membrane staining), it was more pronounced when two accessory elements of the cassette coding for an i-spanin and an o-spanin were included in the overexpression construct. The pore-forming function of the putative holin, HolY, was demonstrated by complementation of the lysis defect of a phage λ S holin mutant. In experiments performed with membrane preparations, ElyY exhibited high specificity for W22703 peptidoglycan, with a cleavage activity resembling that of lysozyme. Although the functionality of the lysis cassette from Tc-PAIYewas demonstrated in this study, its biological role remains to be elucidated.IMPORTANCEThe knowledge of how pathogens survive in the environment is pivotal for our understanding of bacterial virulence. The insecticidal and nematocidal activity ofYersiniaspp., by which the bacteria gain access to nutrients and thus improve their environmental fitness, is conferred by the toxin complex (Tc) encoded on a highly conserved pathogenicity island termed Tc-PAIYe. While the regulators and the toxin subunits of the island had been characterized in some detail, the role of phage-related genes within the island remained to be elucidated. Here, we demonstrate that this cassette encodes a holin, an endolysin, and two spanins that, at least upon overexpression, lyseYersiniastrains.

The low-resolution 3D-structure of porin, a channel-forming protein in E. coliouter membranes

1983 ◽  
Vol 41 ◽  
pp. 440-441
Author(s):  
A. Engel ◽  
D.L. Dorset ◽  
A. Massalski ◽  
J.P. Rosenbusch
Keyword(s):  
Crystal Packing ◽  
3D Structure ◽  
Gram Negative Bacteria ◽  
Protein Ratio ◽  
Crystal Forms ◽  
Large Molecules ◽  
Functional Studies ◽  
Voltage Dependent ◽  
Planar Membranes ◽  
Hexagonal Arrays

Porins represent a group of channel forming proteins that facilitate diffusion of small solutes across the outer membrane of Gram-negative bacteria, while excluding large molecules (>650 Da). Planar membranes reconstituted from purified matrix porin (OmpF protein) trimers and phospholipids have allowed quantitative functional studies of the voltage-dependent channels and revealed concerted activation of triplets. Under the same reconstitution conditions but using high protein concentrations porin aggregated to 2D lattices suitable for electron microscopy and image processing. Depending on the lipid-to- protein ratio three different crystal packing arrangements were observed: a large (a = 93 Å) and a small (a = 79 Å) hexagonal and a rectangular (a = 79 Å b = 139 Å) form with p3 symmetry for the hexagonal arrays. In all crystal forms distinct stain filled triplet indentations could be seen and were found to be morphologically identical within a resolution of (22 Å). It is tempting to correlate stain triplets with triple channels, but the proof of this hypothesis requires an analysis of the structure in 3 dimensions.

3-D optical transfer in excitation field synthesis microscopes

1995 ◽  
Vol 53 ◽  
pp. 64-65
Author(s):  
Vijay Krishnamurthi ◽  
Brent Bailey ◽  
Frederick Lanni
Keyword(s):  
Standing Wave ◽  
Spatial Information ◽  
3D Structure ◽  
Complete Recovery ◽  
Weighting Factor ◽  
Optical Transfer Function ◽  
Excitation Field ◽  
Optical Transfer ◽  
Set Up ◽  
Focus Plane

Excitation field synthesis (EFS) refers to the use of an interference optical system in a direct-imaging microscope to improve 3D resolution by axially-selective excitation of fluorescence within a specimen. The excitation field can be thought of as a weighting factor for the point-spread function (PSF) of the microscope, so that the optical transfer function (OTF) gets expanded by convolution with the Fourier transform of the field intensity. The simplest EFS system is the standing-wave fluorescence microscope, in which an axially-periodic excitation field is set up through the specimen by interference of a pair of collimated, coherent, s-polarized beams that enter the specimen from opposite sides at matching angles. In this case, spatial information about the object is recovered in the central OTF passband, plus two symmetric, axially-shifted sidebands. Gaps between these bands represent "lost" information about the 3D structure of the object. Because the sideband shift is equal to the spatial frequency of the standing-wave (SW) field, more complete recovery of information is possible by superposition of fields having different periods. When all of the fields have an antinode at a common plane (set to be coincident with the in-focus plane), the "synthesized" field is peaked in a narrow infocus zone.

Methods for three-dimensional reconstruction of cellular components within a thick section

1986 ◽  
Vol 44 ◽  
pp. 18-21
Author(s):  
J. Frank ◽  
B. F. McEwen ◽  
M. Radermacher ◽  
C. L. Rieder
Keyword(s):  
Tilt Angle ◽  
Tomographic Reconstruction ◽  
3D Structure ◽  
Three Dimensional ◽  
Thick Section ◽  
Three Dimensional Reconstruction ◽  
Axis Ratio ◽  
Dimensional Reconstruction ◽  
Cellular Components

The tomographic reconstruction from multiple projections of cellular components, within a thick section, offers a way of visualizing and quantifying their three-dimensional (3D) structure. However, asymmetric objects require as many views from the widest tilt range as possible; otherwise the reconstruction may be uninterpretable. Even if not for geometric obstructions, the increasing pathway of electrons, as the tilt angle is increased, poses the ultimate upper limitation to the projection range. With the maximum tilt angle being fixed, the only way to improve the faithfulness of the reconstruction is by changing the mode of the tilting from single-axis to conical; a point within the object projected with a tilt angle of 60° and a full 360° azimuthal range is then reconstructed as a slightly elliptic (axis ratio 1.2 : 1) sphere.

Assembly of VP26 in herpes simplex virus-1 capsid implicated by 3-D structure of recombinant capsid

1995 ◽  
Vol 53 ◽  
pp. 840-841
Author(s):  
Z. Hong Zhou ◽  
Jing He ◽  
Joanita Jakana ◽  
J. D. Tatman ◽  
Frazer J. Rixon ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
3D Structure ◽  
Structure Study ◽  
Herpes Simplex Virus 1 ◽  
Shell Proteins ◽  
Life Threatening ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

Herpes simplex virus-1 (HSV-1) is a ubiquitous virus which is implicated in diseases ranging from self-curing cold sores to life-threatening infections. The 2500 Å diameter herpes virion is composed of a glycoprotein spike containing, lipid envelope, enclosing a protein layer (the tegument) in which is embedded the capsid (which contains the dsDNA genome). The B-, and A- and C-capsids, representing different morphogenetic stages in HSV-1 infected cells, are composed of 7, and 5 structural proteins respectively. The three capsid types are organized in similar T=16 icosahedral shells with 12 pentons, 150 hexons, and 320 connecting triplexes. Our previous 3D structure study at 26 Å revealed domain features of all these structural components and suggested probable locations for the outer shell proteins, VP5, VP26, VP19c and VP23. VP5 makes up most of both pentons and hexons. VP26 appeared to bind to the VP5 subunit in hexon but not to that in penton.

Cryomicroscopy of crotoxin complex crystals at 400 KV

1989 ◽  
Vol 47 ◽  
pp. 826-827
Author(s):  
Jaap Brink ◽  
Wah Chiu
Keyword(s):  
Signal To Noise Ratio ◽  
3D Structure ◽  
Three Dimensional ◽  
Carbon Films ◽  
Depth Of Field ◽  
Basic Subunit ◽  
Ewald Sphere ◽  
Diffraction Patterns ◽  
Complex Crystals ◽  
Acidic Subunit

The crotoxin complex is a potent neurotoxin composed of a basic subunit (Mr = 12,000) and an acidic subunit (M = 10,000). The basic subunit possesses phospholipase activity whereas the acidic subunit shows no enzymatic activity at all. The complex's toxocity is expressed both pre- and post-synaptically. The crotoxin complex forms thin crystals suitable for electron crystallography. The crystals diffract up to 0.16 nm in the microscope, whereas images show reflections out to 0.39 nm2. Ultimate goal in this study is to obtain a three-dimensional (3D-) structure map of the protein around 0.3 nm resolution. Use of 100 keV electrons in this is limited; the unit cell's height c of 25.6 nm causes problems associated with multiple scattering, radiation damage, limited depth of field and a more pronounced Ewald sphere curvature. In general, they lead to projections of the unit cell, which at the desired resolution, cannot be interpreted following the weak-phase approximation. Circumventing this problem is possible through the use of 400 keV electrons. Although the overall contrast is lowered due to a smaller scattering cross-section, the signal-to-noise ratio of especially higher order reflections will improve due to a smaller contribution of inelastic scattering. We report here our preliminary results demonstrating the feasability of the data collection procedure at 400 kV.Crystals of crotoxin complex were prepared on carbon-covered holey-carbon films, quench frozen in liquid ethane, inserted into a Gatan 626 holder, transferred into a JEOL 4000EX electron microscope equipped with a pair of anticontaminators operating at −184°C and examined under low-dose conditions. Selected area electron diffraction patterns (EDP's) and images of the crystals were recorded at 400 kV and −167°C with dose levels of 5 and 9.5 electrons/Å, respectively.

Quantitative structure determination of interfaces through Z-contrast imaging and electron energy loss spectroscopy

1996 ◽  
Vol 54 ◽  
pp. 104-105
Author(s):  
S. J. Pennycook ◽  
P. D. Nellist ◽  
N. D. Browning ◽  
P. A. Langjahr ◽  
M. Rühle
Keyword(s):  
Grain Boundaries ◽  
Cuprate Superconductors ◽  
Structure Refinement ◽  
3D Structure ◽  
Real Space ◽  
Contrast Imaging ◽  
Coordination Polyhedra ◽  
Burgers Vector ◽  
Z Contrast ◽  
Contrast Image

The simultaneous use of Z-contrast imaging with parallel detection EELS in the STEM provides a powerful means for determining the atomic structure of grain boundaries. The incoherent Z-contrast image of the high atomic number columns can be directly inverted to their real space arrangement, without the use of preconceived structure models. Positions and intensities may be accurately quantified through a maximum entropy analysis. Light elements that are not visible in the Z-contrast image can be studied through EELS; their coordination polyhedra determined from the spectral fine structure. It even appears feasible to contemplate 3D structure refinement through multiple scattering calculations.The power of this approach is illustrated by the recent study of a series of SrTiC>3 bicrystals, which has provided significant insight into some of the basic issues of grain boundaries in ceramics. Figure 1 shows the structural units deduced from a set of 24°, 36° and 65° symmetric boundaries, and 24° and 45° asymmetric boundaries. It can be seen that apart from unit cells and fragments from the perfect crystal, only three units are needed to construct any arbitrary tilt boundary. For symmetric boundaries, only two units are required, each having the same Burgers, vector of a<100>. Both units are pentagons, on either the Sr or Ti sublattice, and both contain two columns of the other sublattice, imaging in positions too close for the atoms in each column to be coplanar. Each column was therefore assumed to be half full, with the pair forming a single zig-zag column. For asymmetric boundaries, crystal geometry requires two types of dislocations; the additional unit was found to have a Burgers’ vector of a<110>. Such a unit is a larger source of strain, and is especially important to the transport characteristics of cuprate superconductors. These zig-zag columns avoid the problem of like-ion repulsion; they have also been seen in TiO2 and YBa2Cu3O7-x and may be a general feature of ionic materials.

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