Biomedical subjects

Kenneth H Downing

Publications and source records attributed to Kenneth H Downing.

12 recordsLinked to original sources

PubMed2006

Design of a microfabricated, two-electrode phase-contrast element suitable for electron microscopy.

A miniature electrostatic element has been designed to selectively apply a 90 degrees phase shift to the unscattered beam in the back focal plane of the objective lens, in order to realize Zernike-type, in-focus phase contrast in an electron microscope. The design involves a cylindrically shaped, biased-voltage electrode, which is surrounded by a concentric grounded electrode. Electrostatic calculations have been used to determine that the fringing fields in the region of the scattered electron beams will cause a negligible phase shift as long as the ratio of electrode length to the transverse feature size is greater than 5:1. Unlike the planar, three-electrode einzel lens originally proposed by Boersch for the same purpose, this new design does not require insulating layers to separate the biased and grounded electrodes, and it can thus be produced by a very simple microfabrication process. Scanning electron microscope images confirm that mechanically robust devices with feature sizes of approximately 1 microm can be easily fabricated. Preliminary experimental images demonstrate that these devices do apply a 90 degrees phase shift between the scattered and unscattered electrons, as expected.

Journal Article↗

PubMed2006

Stroboscopic image capture: reducing the dose per frame by a factor of 30 does not prevent beam-induced specimen movement in paraffin.

Beam-induced specimen movement may be the major factor that limits the quality of high-resolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser [Ultramicroscopy 16 (1985) 139-150], which we refer to here as "stroboscopic image capture", is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure--and possibly the amount of beam-induced movement--per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range Charge-coupled device (CCD) camera for image recording and a liquid N2-cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F(image)(g)/F(image)(0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.

Electrons↗

PubMed2006

Molecular architecture of axonemal microtubule doublets revealed by cryo-electron tomography.

The axoneme, which forms the core of eukaryotic flagella and cilia, is one of the largest macromolecular machines, with a structure that is largely conserved from protists to mammals. Microtubule doublets are structural components of axonemes that contain a number of proteins besides tubulin, and are usually found in arrays of nine doublets arranged around two singlet microtubules. Coordinated sliding of adjacent doublets, which involves a host of other proteins in the axoneme, produces periodic beating movements of the axoneme. We have obtained a three-dimensional density map of intact microtubule doublets using cryo-electron tomography and image averaging. Our map, with a resolution of about 3 nm, provides insights into locations of particular proteins within the doublets and the structural features of the doublets that define their mechanical properties. We identify likely candidates for several of these non-tubulin components of the doublets. This work offers insight on how tubulin protofilaments and accessory proteins attach together to form the doublets and provides a structural basis for understanding doublet function in axonemes.

Animals↗

PubMed2006

Oligomerization states of the association domain and the holoenyzme of Ca2+/CaM kinase II.

Ca2+/calmodulin activated protein kinase II (CaMKII) is an oligomeric protein kinase with a unique holoenyzme architecture. The subunits of CaMKII are bound together into the holoenzyme by the association domain, a C-terminal region of approximately 140 residues in the CaMKII polypeptide. Single particle analyses of electron micrographs have suggested previously that the holoenyzme forms a dodecamer that contains two stacked 6-fold symmetric rings. In contrast, a recent crystal structure of the isolated association domain of mouse CaMKIIalpha has revealed a tetradecameric assembly with two stacked 7-fold symmetric rings. In this study, we have determined the crystal structure of the Caenorhabditis elegans CaMKII association domain and it too forms a tetradecamer. We also show by electron microscopy that in its fully assembled form the CaMKII holoenzyme is a dodecamer but without the kinase domains, either from expression of the isolated association domain in bacteria or following their removal by proteolysis, the association domains form a tetradecamer. We speculate that the holoenzyme is held in its 6-fold symmetric state by the interactions of the N-terminal approximately 1-335 residues and that the removal of this region allows the association domain to convert into a more stable 7-fold symmetric form.

Animals↗

PubMed2005

Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography.

Electron tomography is currently the only method that allows the direct three-dimensional visualization of macromolecules in an unperturbed cellular context. In principle, tomography should enable the identification and localization of the major macromolecular complexes within intact bacteria, embedded in amorphous ice. In an effort to optimize conditions for recording data that would bring us close to the theoretical limits, we present here a comparison of the dose tolerance of Caulobacter crescentus cells embedded in amorphous ice at liquid helium versus liquid nitrogen temperature. The inner and outer cell membranes, and the periodic structure of the S-layer of this Gram-negative bacterium provide ideal features to monitor changes in contrast and order as a function of dose. The loss of order in the S-layer occurs at comparable doses at helium and nitrogen temperatures. Macroscopic bubbling within the cell and the plastic support develops at both temperatures, but more slowly at helium temperature. The texture of the bubbles is finer in initial stages at helium temperature, giving an impression of contrast reversal in some parts of the specimen. Bubbles evolve differently in different organelles, presumably a consequence of their different chemical composition and mechanical properties. Finally, the amorphous ice "flows" at helium temperature, causing changes in the relative positions of markers within the specimen and distorting the cells. We conclude that for cryo-electron tomography of whole cells liquid nitrogen temperature provides better overall data quality.

Animals↗

PubMed2005

High-throughput film-densitometry: an efficient approach to generate large data sets.

A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to "near-to-perfect" performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.

Automation↗

PubMed2005

Distinct constrictive processes, separated in time and space, divide caulobacter inner and outer membranes.

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.

Bacterial Outer Membrane Proteins↗

PubMed2004

The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography.

The structure of epothilone A, bound to alpha,beta-tubulin in zinc-stabilized sheets, was determined by a combination of electron crystallography at 2.89 angstrom resolution and nuclear magnetic resonance-based conformational analysis. The complex explains both the broad-based epothilone structure-activity relationship and the known mutational resistance profile. Comparison with Taxol shows that the longstanding expectation of a common pharmacophore is not met, because each ligand exploits the tubulin-binding pocket in a unique and independent manner.

Binding Sites↗

PubMed2004

Electron microscopy of biological macromolecules: bridging the gap between what physics allows and what we currently can get.

The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Because specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over the holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at 0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25%-35% of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.

Crystallization↗

PubMed2003

Meiosis-specific failure of cell cycle progression in fission yeast by mutation of a conserved beta-tubulin residue.

The microtubule cytoskeleton is involved in regulation of cell morphology, differentiation, and cell cycle progression. Precisely controlled dynamic properties are required for these microtubule functions. To better understand how tubulin's dynamics are embedded in its primary sequence, we investigated in vivo the consequences of altering a single, highly conserved residue in beta-tubulin that lies at the interface between two structural domains. The residue differs between the cold-adapted Antarctic fish and temperate animals in a manner that suggests a role in microtubule stability. Fungi, like the Antarctic fish, have a phenylalanine in this position, whereas essentially all other animals have tyrosine. We mutated the corresponding residue in fission yeast to tyrosine. Temperature effects were subtle, but time-lapse microscopy of microtubule dynamics revealed reduced depolymerization rates and increased stability. Mitotic exit signaled by breakdown of the mitotic spindle was delayed. In meiosis, microtubules displayed prolonged contact to the cell cortex during horsetail movement, followed by completion of meiosis I but frequent asymmetric failure of meiosis II spindle formation. Our results indicate that depolymerization dynamics modulated through interdomain motion may be important for regulating a subset of plus-end microtubule complexes in Schizosaccharomyces pombe.

Amino Acid Sequence↗

PubMed2002

Modeling chemical bonding effects for protein electron crystallography: the transferable fragmental electrostatic potential (TFESP) method.

This paper addresses the problem of determining the electrostatic potential of large proteins by the superposition of potentials calculated for small fragments. The use of different atomic and molecular fragments is considered for reproducing the molecular electrostatic potential of different conformations of N-acetylalanine methylamide (NAAMA) with an acceptable degree of error as measured by conventional R factors used in crystallographic structure refinement. Three different divisions of NAAMA are tested, producing fragments that incorporate increasingly more complete descriptions of molecular bonding with diminishing accuracy in geometric fit to the parent molecule: single atoms in molecules, bonded atoms in molecules and selected functional groups, such as the backbone peptide moiety, or the alpha-carbon, beta-carbon and their associated H atoms. In the resolution range 2.5-25A, the fairly straightforward use of single atoms in molecules reduces the calculated R factors by 5-15% over a free-atom superposition. No significant further improvement was found at the lowest resolutions with a superposition of single bonds in molecules and R factors were found to degrade with larger fragments at higher resolutions because of poor geometry fits to the atoms of the parent molecule. Because the potential distribution even for single atoms depends on the environment, the best accuracy will be obtained by using a library of fragment potentials calculated for each type of atom as a function of important protein conformations.

Algorithms↗

PubMed2002

Microtubule structure at 8 A resolution.

We have obtained a 3D reconstruction of intact microtubules, using cryoelectron microscopy and image processing, at a resolution of about 8 A, sufficient to resolve much of the secondary structure. The level of detail in the map allows docking of the tubulin structure previously determined by electron crystallography, with very strong constraints, providing several important insights not previously available through docking tubulin into lower-resolution maps. This work provides an improved picture of the interactions between adjacent protofilaments, which are responsible for microtubule stability, and also suggests that some structural features are different in microtubules from those in the zinc sheets with which the tubulin structure was determined.

Cryoelectron Microscopy↗