Titelbild: Photochemical Electrocyclization of Poly(phenylacetylene)s: Unwinding Helices to Elucidate their 3D Structure in Solution (Angew. Chem. 15/2021)

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.

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3-D optical transfer in excitation field synthesis microscopes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136696 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141871 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140579 ◽

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.

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Cryomicroscopy of crotoxin complex crystals at 400 KV

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156109 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162983 ◽

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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