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Biomedical subjects

M B Sherman

Publications and source records attributed to M B Sherman.

At least 19 recordsLinked to original sources
PubMed1999

Scaling structure factor amplitudes in electron cryomicroscopy using X-Ray solution scattering.

The structure factors derived from electron cryomicroscopic images are modified by the contrast transfer function of the microscope's objective lens and other influences. The phases of the structure factors can be corrected in a straightforward way when the positions of the contrast transfer function rings are determined. However, corrected amplitudes are also essential to yield an accurate distribution of mass in the reconstruction. The correct scale factors for the amplitudes are difficult to evaluate for data that are merged from many different micrographs. We opt to use X-ray solution scattering intensity from a concentrated suspension of the specimen to correct the amplitudes of the spherically averaged structure factors. When this approach is applied to the three-dimensional image data of ice-embedded acrosomal bundles, the core of a filament in a three-dimensional reconstruction of the acrosomal bundle becomes denser and matches more closely the outer density ascribed to scruin.

Acrosome↗
PubMed1999

The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle.

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 microm process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actin-scruin helix that is azimuthally modulated by the influence of the interactions of a filament with its neighbors. There are a variety of density connections with neighboring filaments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three interfilament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Acrosome↗
PubMed1999

Three-dimensional structure of low density lipoproteins by electron cryomicroscopy.

Human low density lipoproteins (LDL) are the major cholesterol carriers in the blood. Elevated concentration of LDL is a major risk factor for atherosclerotic disease. Purified LDL particles appear heterogeneous in images obtained with a 400-kV electron cryomicroscope. Using multivariate statistical and cluster analyses, an ensemble of randomly oriented particle images has been subdivided into homogeneous subpopulations, and the largest subset was used for three-dimensional reconstruction. In contrast to the general belief that below the lipid phase-transition temperature (30 degrees C) LDL are quasi-spherical microemulsion particles with a radially layered core-shell organization, our three-dimensional map shows that LDL have a well-defined and stable organization. Particles consist of a higher-density outer shell and lower-density inner lamellae-like layers that divide the core into compartments. The outer shell consists of apolipoprotein B-100, phospholipids, and some free cholesterol.

Cryoelectron Microscopy↗
PubMed1999

High-resolution electron cryomicroscopy of macromolecular assemblies.

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.

Actin Cytoskeleton↗
PubMed1998

Evaluation of charging on macromolecules in electron cryomicroscopy.

We describe procedures to assess charging of biological specimens under electron irradiation in an electron cryomicroscope. Charging can be observed by an expansion of the illuminating beam, blurring of electron diffraction patterns and by beam "footprints" on the specimen. Discharging can also be seen in the defocused electron diffraction mode. We investigated the influence of a variety of factors on the magnitude and visibility of charging. A reduction of charging is noticed when part of the adjacent carbon film is included in the irradiated specimen area.

Catalase↗
PubMed1998

Multivariate analysis of single unit cells in electron crystallography.

High-resolution electron cryomicroscopy of two-dimensional protein crystals is associated with extremely noisy raw data in which even the crystal lattice often cannot be discerned. Correlation averaging procedures, aimed at calculating the total average of all unit cells of crystals in order to reduce noise, are now used routinely in electron crystallography. Multivariate statistical analysis (MSA) may be used for finding not only the average structure but also for quantifying the systematic departures from that average within the population of individual unit cells. We show that the MSA approach is applicable to single unit-cell images in the low-dose (< 10 electrons/A2), high-resolution (< 5 A) realm using 400 keV electron spot-scan images of ice-embedded gp32*I protein crystals. Our feasibility study opens a pathway toward exploiting these naturally occurring variations on the unit-cell theme in order to achieve higher-resolution three-dimensional reconstruction results, or to better understand the dynamic behaviour of molecules within two-dimensional crystals. We explain how single unit-cell images can be processed and classified into homogeneous groups, and we review how the results of such discriminate averaging may subsequently be exploited within the context of conventional "h, k"-space electron crystallographic approaches. Variations among the individual unit cells may thus be one of the most significant resolution-limiting factors currently experienced in electron crystallography. The quantitative assessment and exploitation of such variations may lead to an increased performance of electron crystallographic procedures.

Bacteriophage T4↗
PubMed1998

Reduction of charging in protein electron cryomicroscopy.

Charging causes a loss of resolution in electron cryomicroscopy with biological specimens prepared without a continuous carbon support film. Thin conductive films were deposited onto catalase crystals prepared across holes using ion-beam sputtering and thermal evaporation and evaluated for the effectiveness of charge reduction. Deposits applied by ion-beam sputtering reduced charging but concurrently resulted in structural damage. Coatings applied by thermal evaporation also reduced charging, and preserved the specimen structure beyond 5 A resolution as judged from electron diffraction patterns and images of glucose-embedded catalase crystals tilted to 45 degrees in the microscope. This study demonstrates for the first time the feasibility of obtaining high-resolution data from unstained, unsupported protein crystals with a conductive surface coating.

Catalase↗
PubMed1997

A strategy for electron tomographic data collection and crystallographic reconstruction of biological bundles.

Structures of highly ordered biological bundles have unique features which call for special experimental and computational methods in electron cryomicroscopy. They can be considered as three-dimensional quasi-crystals and reconstructed using a crystallographic approach. However, they are neither "infinitely" large with respect to the borders of the bundle, nor are they a single unit cell in thickness along the viewing direction. Also, because of their shape, bundles do not generally have a preferred azimuthal orientation, which poses challenges for orientation estimation and refinement. We developed a strategy for recording and processing electron cryomicroscopic images that differs from classical two-dimensional crystalline reconstruction techniques. These developments allowed us to merge data from tomographic tilt series of ice-embedded acrosomal bundles. The goal is to determine accurately amplitudes and phases at the diffraction maxima in terms of hkl indices, and compute a three-dimensional map from the diffraction data.

Acrosome↗
PubMed1997

Reliability of phases retrieved from 400-kV spot-scan images of purple membranes acquired on a slow-scan CCD camera.

Glucose-embedded purple membranes were used as a test specimen to evaluate the reliability of phases retrieved from 400-kV spot-scan images acquired on a 1024 x 1024 slow-scan CCD camera. This specimen was chosen because it represents a broad class of low-contrast radiation-sensitive biological objects and its structure is well established. The amplitudes of computed reflections from these images were strongly damped by the modulation transfer function of the camera. Nevertheless, their phases on average were < 12 degrees different from the reference data of Henderson et al. (1986), Ultramicroscopy, 19, 147-178, up to 8.8 A resolution, which corresponds to 0.8 of the Nyquist frequency of the camera.

Glucose↗
PubMed1996

Performance of a slow-scan CCD camera for macromolecular imaging in a 400 kV electron cryomicroscope.

The feasibility and limitations of a 1024 x 1024 slow-scan charge-coupled device (CCD) camera were evaluated for imaging in a 400kV electron cryomicroscope. Catalase crystals and amorphous carbon film were used as test specimens. Using catalase crystals, it was found that the finite (24 microns) pixel size of the slow-scan CCD camera governs the ultimate resolution in the acquired images. For instance, spot-scan images of ice-embedded catalase crystals showed resolutions of 8 A and 4 A at effective magnifications of 67,000 x and 132,000 x, respectively. Using an amorphous carbon film, the damping effect of the modulation transfer function (MTF) of the slow-scan CCD camera on the specimen's Fourier spectrum relative to that of the photographic film was evaluated. The MTF of the slow-scan CCD camera fell off more rapidly compared to that of the photographic film and reached the value of 0.2 at the Nyquist frequency. Despite this attenuation, the signal-to-noise ratio of the CCD data, as determined from reflections of negatively-stained catalase crystals, was found to decrease to approximately 50% of that of photographic film data. The phases computed from images of the same negatively-stained catalase crystals recorded consecutively on both the slow-scan CCD camera and photographic film were found to be comparable to each other within 12 degrees. Ways of minimizing the effect of the MTF of the slow-scan CCD camera on the acquired images are also presented.

Animals↗
PubMed1995

Electron cryomicroscopy and angular reconstitution used to visualize the skeletal muscle calcium release channel.

We exploit the random orientations of ice-embedded molecules imaged in an electron cryomicroscope to determine the three-dimensional structure of the Ca(2+)-release channel from the sarcoplasmic reticulum (SR) in its closed state, without tilting the specimen holder. Our new reconstruction approach includes an exhaustive search of all different characteristic projection images in the micrographs and the assignment of Euler angle orientations to these views. The 30 A map implied reveals a structure in which the transmembrane region exhibits no apparent opening on the SR lumen side. The extended cytoplasmic region has a hollow appearance and consists, in each monomer, of a clamp-shaped and a handle-shaped domain.

Animals↗
PubMed1991

Three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina.

The three-dimensional structure of the nickel-containing hydrogenase from Thiocapsa roseopersicina has been determined at a resolution of 2 nm in the plane and 4 nm in the vertical direction by electron microscopy and computerized image processing on microcrystals of the enzyme. The enzyme forms a large ring-shaped complex containing six each of the large (62-kDa) and small (26-kDa) subunits. The complex is very open, with six well-separated dumbbell-shaped masses surrounding a large cylindrical hole. Each dumbbell is interpreted as consisting of one large and one small subunit.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Negative staining of proteins.

Negative staining, some closely related alternative preparation techniques and radiation stability are considered. An attempt is made to clarify the mechanism of action and ultimate resolution limit of negative staining. The results of electron diffraction investigation of thermitase microcrystals embedded in glucose and glucose + stains are presented. It is shown that at doses not exceeding 10 electrons/nm2 electron diffraction from thermitase crystals demonstrate diffraction fields up to 0.2 nm. When adding heavy-atom salts to glucose or using negative staining, the relative intensities of reflections change and electron diffraction patterns for every type of heavy-atom additive (or negative stain) have their specific features. Such characteristic changes of reflection intensities indicate specific interaction of these additives (or stains) with the object. In the case of electron diffraction from the crystals stained using the routine negative staining technique the ordering was preserved down to 0.4-0.5 nm. Increasing the dose up to the normal value results in fading of distant reflections. Thus, negative staining with radiation doses less than the critical one could yield resolution down to 0.4 nm. Yet, the structure may change due to interaction with the stain. Nevertheless, the possibility that such resolution could be obtained for a limited number of objects should not be excluded. Some examples of the application of negative staining for investigation of quaternary and domain structure of proteins (nitrogenase, glutamine synthetase, mitochondrial ATP-synthase, membrane monooxygenase enzymes), tubular and two-dimensional protein crystals (catalase, phosphorylase, HWV protein, hydrogenase), as well as ribosomes and bacteriophages are given in the review.

Animals↗
PubMed1987

Effect of early exposure to family medicine on students' attitudes toward the specialty.

In the study reported here, the authors examined the influence of clinical experiences of students on their choice of family medicine as a specialty. Attitudes toward family medicine were investigated among 172 first-year and 174 second-year students at the University of Minnesota Medical School--Minneapolis. Neither early exposure to role models in family medicine nor the order in which specialty clerkships were taken had a significant effect on the students' choice of family medicine as a specialty.

Attitude↗
PubMed1981

On the negative straining of the protein crystal structure.

It has been shown that in some cases negative staining reveals structure with details down to 0.4 nm in size, the nature of protein playing a key role. Various stains seem to interact with different parts of a thermitase (a serine protease) molecule, which results in intensity changes in electron diffraction patterns.

Crystallization↗