Biomedical subjects

R M Glaeser

Publications and source records attributed to R M Glaeser.

At least 37 records · Page 2Linked to original sources

PubMed1992

Assessment of resolution in biological electron crystallography.

The resolution of images or density maps produced by electron microscopy and electron crystallography can be objectively defined in terms of the spatial frequency of the highest resolution diffraction spot, or Fourier coefficient, included in the data processing. In practice, this objective definition of resolution is expected to be too optimistic if the amplitudes of the highest resolution structure factors are too weak, if the population of high resolution reflections is too sparse, or if the signal-to-noise ratio of the high resolution data is too low. Calculated examples are presented here which illustrate how the apparent resolution in images of a membrane protein, bacteriorhodopsin, can be reduced from a nominal value of 3.5 A by weak amplitudes, sparse data or high noise levels. These calculations provide concrete examples which can serve as a guide when estimating whether the objective definition of image resolution is likely to correspond to a practical, structurally useful estimate of image resolution.

Bacteriorhodopsins↗

PubMed1991

Three-dimensional structure of an invertebrate intercellular communicating junction.

Gap junctions containing extensive, highly ordered crystalline arrays of hexagonally packed connexons have been isolated from the hepatopancreas of the arthropod, Homarus americanus (American lobster). The structure of such junctions has been studied to a resolution of approximately 25 A in three dimensions by electron microscopy of negatively stained specimens. The structure, which has the crystallographic symmetry of the two-sided plane group p6, reveals the connexon as an annular oligomer which projects approximately 30-45 A from the cytoplasmic surface. The stain-filled channel structure appears to be approximately 40-45 A wide in the extracellular region. Projection images of glucose-embedded specimens extend to a resolution of 10 A, and show a strong contrast from the connexon subunits. Overall the structure is quite similar to that of rat liver junctions, except that less stain is seen in the aqueous region of the gap and more surrounding the protrusions of the protein into the cytoplasm.

Animals↗

PubMed1991

What spectroscopy can still tell us about the secondary structure of bacteriorhodopsin.

The recently published model of the structure of bacteriorhodopsin (bR), developed by fitting the peptide chain to a high-resolution, three-dimensional density map, rules out the existence of transmembrane beta-sheet and provides an accurate estimate of the helix content. The precise geometry of the dihedral angles in the helical regions of the polypeptide cannot yet be specified from the diffraction data, however. Published data on the circular dichroism (CD) spectrum between 190 and 240 nm, and the infrared (IR) spectrum in the amide I band suggest that the helical conformation in bR may be, for the most part, a rather unusual one. The precise structural model, which specifies the number of residues in transmembrane helices, can now be used as an additional constraint in seeking models of the helical conformation that are in quantitative agreement with the CD and IR spectroscopic data. Further spectroscopic measurements can also be used to determine whether there are changes in the unusual dihedral-angle conformation within the helices during the photocycle.

Bacteriorhodopsins↗

PubMed1991

Interfacial energies and surface-tension forces involved in the preparation of thin, flat crystals of biological macromolecules for high-resolution electron microscopy.

It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1 degree, and perhaps less than 0.2 degrees, over areas as large as 1 micron2 or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.

Adsorption↗

PubMed1989

High-voltage electron diffraction from bacteriorhodopsin (purple membrane) is measurably dynamical.

Electron diffraction patterns of 45 A thick two-dimensional crystalline arrays of a cell membrane protein, bacteriorhodopsin, have been recorded at two electron voltages, namely 20 and 120 kV. Significant intensity differences are observed for Friedel mates at 20 kV, but deviations from Friedel symmetry are quite small at 120 kV. It does not seem likely that the measured Friedel differences can be accounted for by complex atomic structure factors, by curvature of the Ewald sphere, or by effects that might occur as a result of inelastic scattering (absorption). It is therefore concluded that dynamical diffraction within the single molecular layer of these crystals is responsible for the observed Friedel differences. The results are useful in estimating the maximum specimen thickness for which the kinematic approximation may be safely used in electron crystallography of biological macromolecules at the usual electron voltage of 100 kV, or even at higher voltages. The results show that the Friedel differences are independent of resolution and this opens up the possibility that dynamical effects occurring at lower voltages might be used to phase higher-voltage kinematic diffraction intensities.

Bacteriorhodopsins↗

PubMed1989

Three-dimensional reconstructions from incomplete data: interpretability of density maps at "atomic" resolution.

Three dimensional data collection in electron microscopy is normally limited to a range of tilt angles that is less than +/- 90 degrees. Thus, even under the best conditions, experimental values of the structure factors will be missing within a solid cone in reciprocal space. Previous work has already shown that the missing cone of data can produce serious artifacts in three-dimensional density maps at modest resolution, for example approximately 15A. We now report, however, that a missing cone as large as +/- 30 degrees appears to have no serious effect on the three-dimensional density map of a protein at 3.6 A resolution, and we attribute this result to the fact that the electron density features are quite well separated from one another at that resolution. The map calculated with a +/- 30 degree missing cone is, furthermore, no more sensitive to noise (error) than is the full (isotropic) Fourier map. This result does not seem to be unreasonable in view of the fact that less than 14% of the data (i.e., signal) is lost due to the missing cone. Our numerical simulations therefore indicate that there should be no difficulty in interpreting high-resolution Fourier maps that can be produced with data that fall within realistic estimates of achievable resolution, tilt angles, and experimental error.

Electronic Data Processing↗

PubMed1988

Validity domain of the weak-phase-object approximation for electron diffraction of thin protein crystals.

The domain of validity of the weak-phase-object (WPO) approximation is evaluated for high-energy electrons (100 keV, 500 keV and 1 MeV) scattered by crystalline biological macromolecules. Cytochrome b5 is used as an example in which calculated dynamical diffraction intensities are used to simulate observed diffraction intensities which are then compared with intensities calculated by the weak-phase-object approximation. Three criteria of validity are used, namely the crystallographic residual (R value), the interpretability of difference Patterson maps, and the results of phasing by the heavy-atom isomorphous replacement method. The present calculations indicate that the error associated with the WPO approximation is quite acceptable up to a specimen thickness of 200 A for 100 keV electrons, which is two to four times the thickness limit for crystalline organic structures with much smaller unit-cell dimensions. An equally acceptable thickness limit at 500 keV and 1MeV is about 300-350 A.

Crystallography↗

PubMed1987

Difference Fourier analysis of "surface features" of bacteriorhodopsin using glucose-embedded and frozen-hydrated purple membrane.

A difference Fourier map of the projected structure of bacteriorhodopsin has been synthesized from electron diffraction amplitudes collected from membranes prepared in the glucose-embedded state and the frozen-hydrated state. Phases of a recently published data set for glucose-embedded specimens were used for the difference Fourier map. Moderate resolution (9 A) and high resolution (4.25 A) maps both indicate that glucose is exchangeable for water in the region of the map corresponding to the lipid regions. We interpret this as indicating that there is a small surface depression in this region of the structure. The depth of this feature is estimated to be 1/6 the thickness of the protein region in the membrane. The data obtained in this study rules out the existence of an aqueous transmembrane channel, the dimensions of which are large enough to allow free exchange of glucose for water. Several new features are also observed in the protein region of the membrane. These features are probably due to segments of the polypeptide at the aqueous interface that are well ordered in frozen-hydrated specimens but not in glucose-embedded specimens. Candidate structures for the origin of these features are extensions of the helices, or linker regions connecting the helices.

Bacteriorhodopsins↗

PubMed1986

Improvement in high resolution image quality of radiation-sensitive specimens achieved with reduced spot size of the electron beam.

High resolution images of thin paraffin crystals and of purple membrane (i.e., crystalline bacteriorhodopsin) have been recorded with illumination spots that are confined to a size of 1000 to 2000 A. Images recorded in this way show three to five times greater contrast than do images which are recorded with more conventional flood-beam (ca. 3 micron diameter) illumination. In addition, optical diffraction patterns of small areas of such images show much better preservation of contrast in all directions than is normally the case. Both effects substantiate the idea that beam-induced movement of radiation-sensitive organic specimens has been a major factor which has caused low dose images to be greatly inferior in their quality compared to electron diffraction patterns of the same type of specimens. In spite of the marked improvement that is obtained, we often observe an unexplained degree of contrast variation within the small illuminated area, demonstrating that even further improvement in the quality of low dose images should be possible.

Bacteriorhodopsins↗

PubMed1986

Morphological observations on the formation and stability of the crystalline arrays in the plasma membrane of Saccharomyces cerevisiae.

Two-dimensional crystalline arrays of freeze-fracture particles are known to occur in abundant quantities in the plasma membrane of stationary state yeast cells. Although these crystalline arrays are seen only infrequently in cells during mid-exponential growth, we now observe that formation of crystalline arrays can be induced in such cells by a "metabolic starvation" protocol. Surprisingly, starvation-induced formation of crystalline patches can be prevented by inhibition of new protein synthesis during the starvation period. The size and quantity of crystalline arrays can be increased by removal of the cell wall prior to starvation. Induction of crystalline arrays in protoplasts has made it possible to investigate the surface morphology of the crystalline particles in isolated membranes as well as at the extracellular surface of intact protoplasts. The stability of isolated crystalline arrays to several detergents has been investigated and conditions have been found that result in improved morphological purity of the isolated crystalline patches.

Cell Cycle↗

PubMed1986

Electron diffraction analysis of the M412 intermediate of bacteriorhodopsin.

High resolution electron diffraction data have been recorded for glucose-embedded purple membrane specimens in which bacteriorhodopsin (bR) has been trapped by cooling slowly to below--100 degrees C under continuous illumination. Thin films (OD approximately 0.7) of glucose-embedded membranes, prepared as a control, showed virtually 100% conversion to the M state, and stacks of such thin film specimens gave very similar x-ray diffraction patterns in the bR568 and the M412 state in most experiments. To be certain that any measured differences in diffraction intensity would be real, two independent sets of electron diffraction intensities were recorded for near-equatorial, i.e. (hkO), reflections. Little correlation was indeed observed between these two sets for delta F values at low resolution (15-5.0 A, 49 reflections), but the correlation coefficient is approximately 0.3 at high resolution (5.0-3.3 A, 218 reflections). Thus, while most of the measured difference is error, the mean delta F and the correlation coefficient can be used to estimate the smaller, true delta F due to structural changes occurring in the M state. The magnitude of this estimated true mean delta F is equal to what would be produced if approximately five to seven nonhydrogen atoms were moved to structurally uncorrelated (i.e., new) positions in the M state. Movements of a few amino acid side chains, and repositioning of atoms of the retinal group and the associated lysine side chain after trans-cis isomerization, are the most probable causes of the observed intensity changes in the M state. The difference Fourier map, calculated in projection at 3.5-A resolution, shows only very small peaks, the largest of which are confined, however, to the region of the protein.

Bacteriorhodopsins↗

PubMed1986

Three-dimensional structure of the surface layer protein of Aquaspirillum serpens VHA determined by electron crystallography.

The three-dimensional structure of the protein which forms the S layer of Aquaspirillum serpens strain VHA has been determined by electron microscopy. Structures have been reconstructed to a resolution of about 1.6 nm for single-layered specimens and about 4 nm for two-layered specimens. The structure, which has hexagonal symmetry, consists of a core in the shape of a cup, with six projections arising from the rim of the cup to join adjacent subunits at the threefold symmetry axes. The model is consistent with edge views of the S layer which have been obtained in this and other work. It is now clear from this work and from three-dimensional reconstructions of other bacterial S layers that a wide diversity exists in the morphology of surface layers.

Bacterial Proteins↗

PubMed1985

Absorption flattening in the circular dichroism spectra of small membrane fragments.

The inhomogeneous distribution of chromophore occurring in a particulate suspension can result in a reduction in the apparent molar ellipticity recorded in circular dichroism (CD) spectra. The possibility of such a systematic error has often been a matter of concern when CD spectra of cell membrane proteins are recorded. The recent publication of CD spectra for bacteriorhodopsin in native and sonicated membranes, in detergent-solubilized form, and reconstituted into small unilamellar vesicles [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673] gives a unique opportunity to apply the theoretical analysis of Gordon and Holzwarth [Gordon, D. J., & Holzwarth, G. (1971) Arch. Biochem. Biophys. 142, 481-488] so as to provide a definitive answer to the question of whether absorption flattening is significant for membrane particles. We show here that the data of Mao and Wallace can be combined with the theoretical analysis of Gordon and Holzwarth to rule out significant absorption flattening effects over the range 200-240 nm for submicrometer-sized membranes. In addition, the results show that absorption flattening can be disregarded even at 190 nm for membranous material in the size range below 100 nm. The demonstration that there are no major flattening effects in the CD spectra of bacteriorhodopsin, particularly in the region of 200-240 nm, means that the experimental spectra are incompatible with the proposal that this transmembrane protein contains seven transmembrane helices.

Bacteriorhodopsins↗

PubMed1985

Structural comparison of native and deoxycholate-treated purple membrane.

Lipid-depleted purple membrane prepared by extraction with deoxycholate has been compared with the native structure. X-ray and electron diffraction photographs show a reduction in cell dimension from 62.4 to 57.3 A, and a substantial change in the distribution of diffraction intensity compared with the native specimens. Low-dose electron microscopy has been used to obtain a projected density map of lipid-depleted membranes. The projected structure shows that the deoxycholate treatment removes a boundary layer of lipid, which in the native form separates adjacent trimers of bacteriorhodopsin. The map also provides an improved estimate of the molecular envelope of the protein. A plausible arrangement for the lipid molecules in both the native and the lipid-depleted membranes is proposed, but the precise positions of individual molecules cannot yet be specified.

Bacteriorhodopsins↗

PubMed1985

Projected structure of the pore-forming OmpC protein from Escherichia coli outer membrane.

A single-projection structure analysis of a bacterial outer membrane protein, OmpC, has been carried out by electron microscopy of frozen hydrated specimens. Two distinct crystal polymorphs have been observed in the frozen-hydrated samples, and projection structures of both forms have been obtained to a resolution of 13.5 A. Preliminary examination of negatively stained samples revealed the expected, trimeric appearance of pores in the OmpC specimens. Electron microscopy of unstained, frozen-hydrated OmpC reveals the trimeric pore structure with equal clarity. In addition, the overall molecular envelope of the protein is readily discerned, and a major lipid-containing domain can also be seen. Because of the small coherent patch size, mosaic disorder, and unpredictable polymorphism of the presently available specimens, three-dimensional reconstruction of frozen-hydrated OmpC has not been carried out.

Bacterial Outer Membrane Proteins↗

PubMed1984

Preparation of frozen-hydrated specimens for high resolution electron microscopy.

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.

Freeze Drying↗

PubMed1983

Quantitative evaluation of high-resolution features in images of negatively stained Tobacco Mosaic Virus.

This study investigates the causes of the apparent differences between the optical diffraction pattern of a micrograph of a Tobacco Mosaic Virus (TMV) particle, the optical diffraction pattern of a ten-fold photographically averaged image, and the computed diffraction pattern of the original micrograph. Peak intensities along the layer lines in the transform of the averaged image appear to be quite unlike those in the diffraction pattern of the original micrograph, and the diffraction intensities for the averaged image extend to unexpectedly high resolution. A carefully controlled, quantitative comparison reveals, however, that the optical diffraction pattern of the original micrograph and that of the ten-fold averaged image are essentially equivalent. Using computer-based image processing, we discovered that the peak intensities on the 6th layer line have values very similar in magnitude to the neighboring noise, in contrast to what was expected from the optical diffraction pattern of the original micrograph. This discrepancy was resolved by recording a series of optical diffraction patterns when the original micrograph was immersed in oil. These patterns revealed the presence of a substantial phase grating effect, which exaggerated the peak intensities on the 6th layer line, causing an erroneous impression that the high resolution features possessed a good signal-to-noise ratio. This study thus reveals some pitfalls and misleading results that can be encountered when using optical diffraction patterns to evaluate image quality.

Computers↗

PubMed1983

Peptide-chain secondary structure of bacteriorhodopsin.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.

Bacteriorhodopsins↗