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A Massalski

Publications and source records attributed to A Massalski.

12 recordsLinked to original sources
PubMed1989

Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy.

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.

Bacterial Outer Membrane Proteins↗
PubMed1984

3D reconstruction from electron micrographs: its potential and practical limitations.

The 3D structure of stain-penetrated transmembrane channels of E. coli outer membrane porin has been reconstructed from electron microscopic projections. The averaged unit cells of the projected hexagonal porin lattices were calculated by correlation averaging. Amplitude and phase values along the continuous lattice lines were obtained from the unit cell transforms, and continuous curves were fitted using either a cubic spline-fit or by convolution with an appropriate sinc function. The reconstructed model shows that the three channels of a porin triplet merge to a single channel while traversing the membrane. This is in excellent agreement with results of functional studies. Some technical difficulties and limitations as well as the potential of the reconstruction process are discussed.

Bacterial Proteins↗
PubMed1983

Two-dimensional crystal packing of matrix porin. A channel forming protein in Escherichia coli outer membranes.

Two-dimensional crystalline porin sheets were obtained by reconstitution of monodisperse protein trimers and phospholipids (dimyristoylphosphatidylcholine) by detergent dialysis, analogous to the reconstitution method used for functional tests (Schindler & Rosenbusch, 1981). Three different packing arrangements were observed: two were hexagonal (with p3 symmetry and lattice constants of 9.3 nm and 7.9 nm), and one rectangular (a = 7.9 nm, b = 13.9 nm). The different crystals could be correlated to phospholipid-to-protein weight ratios of 0.16 to 0.72. At the higher ratio, large hexagonal lattices predominated. Higher lipid ratios did not reveal other crystal forms. The packing arrangement of the large hexagonal form appears very similar to the hexagonal habit of three-dimensional crystal forms (Garavito et al., 1983). The shape of the stain-penetrated triplet indentations appeared conserved in the crystal forms to a resolution of 2.2 nm. The mass distribution between triplets, however, were significantly different. They are likely to correspond primarily to lipids. Mass determinations of unstained porin by scanning transmission electron microscopy showed that unit cells consisted of single trimers. The mass found (100,000 daltons) is in good agreement with the value obtained by sedimentation equilibrium analysis.

Bacterial Proteins↗
PubMed1982

In vivo and in vitro models of demyelinating diseases. V. Comparison of the assembly of mouse hepatitis virus, strain JHM, in two murine cell lines.

The developmental sequence of a neurotropic strain (JHM) of mouse hepatitis virus was examined by transmission electron microscopy and immunocytology. The nucleoprotein core of this coronavirus, which contains RNA of positive polarity and is helical in configuration, becomes incorporated into enveloped particles in the same manner as the nucleocapsids of the orthomyxo- and paramyxoviruses. However, JHM virus is assembled intracellularly by budding at surfaces of smooth membranous vacuoles. A comparison of JHM virus replication in L2 and 17Cl-1 cell lines revealed that L2 cells undergo more rapid cytopathology and cease virus production much sooner than 17Cl-l cells. In L2 cells the accumulation of core material appears to continue after the abrupt cessation of virus assembly. This is evident by the massive cytoplasmic accumulation of structure resembling nucleocapsids, which react with hybridoma antibody to the nucleocapsid antigen as demonstrated by the immunoperoxidase procedure. The current findings are consistent with our previously published demonstration, using cells of neural and other deviation, of the fundamental role of the host cell type in regulating the replication and expression of coronaviruses.

Animals↗
PubMed1976

Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis.

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.

Cell Membrane↗
PubMedDate not supplied

Porin channel triplets merge into single outlets in Escherichia coli outer membranes.

Previous observations on the structural and functional properties of porin, the matrix protein of Escherichia coli, have indicated that the channel-forming trimers span the outer membranes of the bacterial cell, forming a molecular sieve. By using electron microscopy and image reconstruction, we demonstrate here that three channels on the outer surface of the cell merge into a single channel at the periplasmic face. Conductance measurements using conditions under which single activated triplets could be observed led us to conclude that the three individual consecutive closing steps reflect three channels within a single trimeric unit. Statistical analysis of conductance levels revealed that the first relaxation step is distinctly smaller than the two subsequent channel closings. This functional observation can be explained if the channels of porin trimers coalesce.

Bacterial Outer Membrane Proteins↗