Pathways for penetration of iron and negative stain across the protein shell of ferritin: Ultrastructural perspectives

1992 ◽  
Vol 50 (2) ◽  
pp. 1012-1013
Author(s):  
William H. Massover
Keyword(s):  
Outer Shell ◽  
X Ray Diffraction ◽  
Central Cavity ◽  
X Ray ◽  
Subunit Dissociation ◽  
Negative Stain ◽  
Analogous Question ◽  
Protein Shell

Molecules of the metalloprotein, ferritin, have an outer shell comprised of a polymeric assembly of 24 polypeptide subunits (apoferritin). This protein shell encloses a hydrated space, the central cavity, within which up to several thousand iron atoms can be deposited as the biomineral, ferrihydrite. The actual pathway taken by iron moving across the protein shell is not known; an analogous question exists for the demonstrated entrance of negative stains into the central cavity. Intersubunit interstices at the 4-fold and 3-fold symmetry axes have been defined with x-ray diffraction, and were hypothesized to provide a pathway for penetration through the outer shell; however, since these channels are only 4Å in width, they are much too small to allow simple permeation of either hydrated iron or stain ions. A different hypothesis, based on studies of subunit dissociation from highly diluted ferritin, proposes that transient gaps in the protein shell are created by a rapid reversible subunit release and permit the direct passage of large ions into the central cavity.

Tubulin structure at intermediate resolution

1995 ◽  
Vol 53 ◽  
pp. 852-853
Author(s):  
K. H. Downing ◽  
S. G. Wolf ◽  
E. Nogales
Keyword(s):  
High Resolution ◽  
Structural Data ◽  
Therapeutic Drug ◽  
Zinc Ions ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray ◽  
Negative Stain ◽  
Functional Mechanisms ◽  
Tubulin Structure

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.

scholarly journals Visualization of a 21-nm axial periodicity in shadowed keratin filaments and neurofilaments.

1982 ◽  
Vol 94 (3) ◽  
pp. 592-596 ◽  
Author(s):  
L Milam ◽  
H P Erickson
Keyword(s):  
Surface Structure ◽  
Intermediate Filaments ◽  
Outer Shell ◽  
X Ray Diffraction ◽  
X Ray ◽  
Helical Pitch ◽  
Left Handed ◽  
Keratin Filaments ◽  
Rotary Shadowing

Unidirectional and rotary shadowing techniques have been applied in studying the surface structure of two types of intermediate filaments. Keratin filaments and neurofilaments demonstrate a approximately 21-nm axial periodicity which probably indicates the helical pitch of the outer shell of the filament. Analysis of unidirectionally shadowed keratin showed that the helix is left-handed. The observation of a left-handed helix of 21-nm pitch supports the three-stranded protofilament model of Fraser, Macrae, and Suzuki (1976, J. Mol. Biol. 108:435-452), and indicates that keratin filaments probably consist of 10 three-stranded protofilaments surrounding a core of three such protofilaments, as predicted by models based on x-ray diffraction of hard keratin filaments. Neurofilaments do not demonstrate an easily identifiable hand, so their consistency with the model is, as yet, uncertain.

scholarly journals Cross-seeding Between the Functional Amyloidogenic CRES and CRES3 Family Members and their Regulation of Aβ Assembly

2020 ◽  
pp. jbc.RA120.015307
Author(s):  
Hoa Quynh Do ◽  
Aveline Hewetson ◽  
Collin G Borcik ◽  
Mary Catherine Hastert ◽  
Sandra Whelly ◽  
...  
Keyword(s):  
Normal Component ◽  
X Ray Diffraction ◽  
Cellular Mechanisms ◽  
Functional Amyloid ◽  
X Ray ◽  
Negative Stain ◽  
Transmission Electron ◽  
Evolutionarily Conserved ◽  
Functional Amyloids ◽  
Amyloid Assembly

Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-β reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest CRES and CRES3 heterooligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aβ by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aβ assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.

Diameter Determination of Frozen-Hydrated Virions Using Polyoma Virus as a Calibration Standard

1988 ◽  
Vol 46 ◽  
pp. 166-167
Author(s):  
N. H. Olson ◽  
T. S. Baker
Keyword(s):  
Accurate Determination ◽  
Carbon Substrate ◽  
Specimen Preparation ◽  
X Ray Diffraction ◽  
Polystyrene Spheres ◽  
X Ray ◽  
Negative Stain ◽  
Highly Sensitive ◽  
Particle Images

Accurate determination of particle dimensions requires both a reliable measure of the instrumental magnification and reproducible, non-distorting specimen preparation procedures. Typical calibration standards for measuring microscope magnification include replica gratings, polystyrene spheres, and negatively-stained catalase crystals. Polystyrene spheres and catalase crystals may be used as internal standards but both are highly sensitive to beam damage. Calibrations with replica gratings are subject to greater inaccuracies at magnifications exceeding 10,000-20,000 X. Furthermore, for negatively-stained biological specimens, the object of interest as well as the standard (e.g. catalase) are susceptible to significant distortions produced when the stained sample dries on the grid. The stain itself also moves during the initial stages of irradiation.1.2Large discrepancies are often found between diameter measurements from particle images with circular profiles (e.g. spherical viruses) made in the microscope and from those measurements determined by x-ray solution scattering or other x-ray diffraction techniques. Measurements from virions embedded in negative-stain, suspended over holes in a carbon substrate, are typically much lower than the corresponding measurements by x-ray techniques, reflecting a probable shrinkage of virions in the stain.

THE RELATION BETWEEN STRUCTURE AND FUNCTION IN HEMOGLOBINS

1964 ◽  
Vol 42 (6) ◽  
pp. 763-775 ◽  
Author(s):  
Austen Riggs
Keyword(s):  
Structural Changes ◽  
Structure And Function ◽  
Bohr Effect ◽  
X Ray Diffraction ◽  
X Ray ◽  
Subunit Dissociation ◽  
And Function ◽  
Polypeptide Chains ◽  
Protein Structure Data ◽  
Lines Of Evidence

Many lines of evidence indicate that the oxygenation of hemoglobin is accompanied by changes in protein structure. Data on the oxygen equilibria of the hemoglobins from a number of animals are discussed in terms of this evidence. Evidence from studies of some hemoglobins (lamprey, frog and tadpole) indicates a major role for subunit dissociation equilibria in explaining two properties of the oxygen equilibria: heme–heme interaction and the "Bohr effect". The importance of subunit dissociation in mammalian hemoglobins is suggested by the known concentration dependence of the oxygen equilibria. Mammalian hemoglobins are composed of two types of polypeptide chains, α and β. The idea that the α and β subunits have different oxygen equilibria and are affected differently by pH is examined. It is concluded that the β-chains appear to play a major role in the mechanism of the Bohr effect not shared by the α-chains. This conclusion is supported by the structural changes in hemoglobin found to occur upon oxygenation by X-ray diffraction techniques.

A Tetragonal Lattice in Mammalian Lens Junctions

1981 ◽  
Vol 39 ◽  
pp. 632-633
Author(s):  
J.D. Robertson ◽  
G. Zampighi ◽  
S. Simon ◽  
T.J. McIntosh ◽  
M.J. Costello
Keyword(s):  
Freeze Fracture ◽  
X Ray Diffraction ◽  
Major Band ◽  
Direction Parallel ◽  
X Ray ◽  
Negative Stain ◽  
Tetragonal Lattice ◽  
Section Electron ◽  
A Minor ◽  
Ray Patterns

A pure fraction of junctional membranes has been isolated in milligram quantities from bovine lens without detergents. SDS-PAGE analysis shows a major band at 27,000 d and a minor one at 21,000 d. Thin section electron microscopy (EM) of this fraction revealed junctions of two closely apposed membranes with an overall thickness of 135-140 Å with a wavy contour and lengths greater than 10 μm. Negative stain and freeze-fracture (FF) EM (only unetched specimens so far) and X-ray diffraction studies of unfixed and unglycerinated specimens were in agreement regarding the structure of the junctions. They consisted primarily of extensive domains of sub-units arranged in a 66 Å tetragonal lattice. Specimens were spun down centrifugally in a Beem capsule and partially dried for the x-ray diffraction studies. The X-ray patterns contained several reflections which indexed on a tetragonal lattice of 66 Å in the equatorial direction parallel to the plane of the membranes and several bands of a continuous transform in the meridional direction consistent with membrane pairs. No diffraction was seen at 85 Å in the equatorial direction.

Comparison of Simulated Images With Electron Images of Lactic Dehydrogenase

1972 ◽  
Vol 30 ◽  
pp. 602-603
Author(s):  
A. Max Fiskin ◽  
Patricia Mack
Keyword(s):  
Metal Ion ◽  
Quaternary Structure ◽  
Lactic Dehydrogenase ◽  
Rabbit Muscle ◽  
X Ray Diffraction ◽  
X Ray ◽  
Molecular Weights ◽  
Simulation Procedure ◽  
Negative Stain ◽  
Simulated Images

Comparison of negative stain images of glutamate dehydrogenase (GDH) with shadow projections of opaque models has revealed that this approach can be misleading. We have suggested an x-ray simulation procedure in which the influence on the image of disruption of oligomer, finite metal-ion complex size, and depth of the stain layer are simulated under controlled conditions, and have deduced a model of GDH quaternary structure using this procedure.Here the accuracy of procedure suggested above is tested by comparing micrographs of rabbit muscle lactic dehydrogenase (LDH) with simulated images obtained with a model of dogfish LDH constructed in accord with x-ray diffraction data. The dogfish and rabbit muscle LDH's exhibit extensive sequence homology, are each composed of four subunits, and have nearly identical molecular weights. Dogfish LDH exhibits 222 symmetry and the shape of the oligomer approximates a parallelepiped.

scholarly journals Cell Adhesion Molecule L1 in Folded (Horseshoe) and Extended Conformations

2001 ◽  
Vol 12 (6) ◽  
pp. 1765-1773 ◽  
Author(s):  
Gregor Schürmann ◽  
Jeffrey Haspel ◽  
Martin Grumet ◽  
Harold P. Erickson
Keyword(s):  
Electron Microscopy ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
X Ray Diffraction ◽  
X Ray ◽  
Negative Stain ◽  
Cell Adhesion Molecule L1 ◽  
First Time ◽  
Compact Shape

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

1973 ◽  
Vol 31 ◽  
pp. 132-133 ◽  
Author(s):  
R. E. Herfert
Keyword(s):  
Surface Characterization ◽  
Analytical Techniques ◽  
X Ray Diffraction ◽  
Depth Of Penetration ◽  
X Ray ◽  
The Past ◽  
Transmission Electron ◽  
Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

Ribosome Structural Organization

1974 ◽  
Vol 32 ◽  
pp. 332-333
Author(s):  
James A. Lake
Keyword(s):  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Small Subunit ◽  
Structural Studies ◽  
X Ray Diffraction ◽  
Specific Antibodies ◽  
X Ray ◽  
E Coli ◽  
Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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