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K H Downing

Publications and source records attributed to K H Downing.

At least 55 records · Page 3Linked to original sources
PubMed1992

Automatic focus correction for spot-scan imaging of tilted specimens.

The variation in defocus within an image of a highly tilted specimen can be a serious source of artifact. Spot-scan imaging can be combined with dynamic focusing to greatly reduce this range of defocus. A protocol is described for determining the parameters required for the automatic focus compensation during the recording of a spot-scan image. Images of a gold test specimen demonstrate the efficacy of this procedure in extending the area of the image that contains high-quality data. In case the tilt angle or resolution is high enough that the height difference of the specimen within each small illuminated area is larger than the depth of field, the image must be treated to compensate for the focus variation. The same principle is used as was developed for compensation of conventional images of tilted specimens.

Artifacts↗
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↗
PubMed1992

Three-dimensional crystallographic reconstruction for atomic resolution.

Three-dimensional structures have recently been determined by electron crystallography at a resolution high enough to determine atomic arrangements in both protein and mineral specimens. The different nature of these two types of specimens produces some very significant differences in the way data is obtained and processed, although the principles are the same. The sensitivity of proteins to damage by the electron beam limits the signal-to-noise ratio in the image and the resolution to which data can be extracted from the image. A number of constraints, such as the amino acid sequence and the connectivity of atoms within amino acids, can be used in interpreting the limited image data. In materials samples, the relative insensitivity to damage allows obtaining resolution limited only by the microscope. In many samples, dynamical scattering and other non-linear effects limit the information in the image, but this limit can be circumvented by working in very thin areas of the specimen.

Bacteriorhodopsins↗
PubMed1991

Spot-scan imaging in transmission electron microscopy.

The determination of the structure of proteins and other organic materials by transmission electron microscopy is a rapidly developing field. Obtaining high-resolution images of these radiation-sensitive specimens has, until recently, been problematic. The development of spot-scan imaging, in which the electron beam is focused to a spot with a diameter of about 1000 angstroms and moved over the specimen to record the image, has overcome some of the most severe problems, which result from beam-induced motion of the specimen and its image. Elimination of this motion greatly enhances the contrast of high-resolution features of the image and promises a significant increase in the speed with which future structural work can be accomplished.

Bacterial Outer Membrane Proteins↗
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↗
PubMed1990

Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy.

The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 A. The map of the structure has a resolution of 3.5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven alpha-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.

Bacteriorhodopsins↗
PubMed1990

Cold stage design for high resolution electron microscopy of biological materials.

Both the number and range of applications of cryotechniques in transmission electron microscopy are increasing rapidly. In some cases, most notably the determination of protein structure by electron crystallography, progress has been limited by the performance of commercially available cryo stages. We review the design and performance criteria for stages which will be necessary for wide applicability in high resolution studies of biological specimens. The important criteria include an operating temperature below -140 degrees C with a low rate of contamination of the specimen, ability to tilt to 60 degrees, and perhaps most important, good resolution as judged by an effective modulation transfer function of 0.8 at 0.35 nm. Most applications also require an effective cryotransfer system. Up until now, most work in high resolution electron crystallography has been accomplished with laboratory-built stages which meet some, but not all, of these criteria. The availability of cold stages which fully meet criteria will allow the rapid expansion of high resolution studies by electron microscopy in structural biology.

Cryopreservation↗
PubMed1990

Structure of PhoE porin in projection at 3.5 A resolution.

The structure of PhoE porin in projection normal to the membrane plane has been determined to a resolution of about 3.5 A by electron crystallographic techniques. The purified protein was reconstituted with lipid to form two-dimensional crystals. High resolution images and electron diffraction patterns of these specimens embedded in trehalose were recorded to obtain respectively the structure factor phase information and the more accurate values of the amplitude. The projection map shows interesting features that are not seen in the earlier map at 6.5 A. Details of the trimeric ring-like structures in our earlier map are now resolved. Each ring-like structure consists of "beads" with interbead spacings of about 4-6 A. These beads are interpreted as the projections of beta-strands along the strands' axes. At the center of the trimeric structure, there is a low density region that we proposed previously to be the location of lipopolysaccharide. Within each ring-like structure, there are complicated features which may play an important role in the size, selectivity, and stability of the channel.

Bacterial Outer Membrane Proteins↗
PubMed1990

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.

Adsorption↗
PubMed1989

Two-dimensional structure of plant light-harvesting complex at 3.7 A [corrected] resolution by electron crystallography.

The structure of the light-harvesting chlorophyll a/b-protein complex has been determined at 3.7 A resolution in projection by electron diffraction, electron microscopy and image analysis. Diffraction patterns and high-resolution spotscan images of two-dimensional crystals stabilized with tannin were recorded at low temperature. Phases of structure factors were obtained directly by image processing, after correction of the images for lattice distortions, defocus and beam tilt. Amplitudes were measured by electron diffraction. The projection map shows the detailed structure of the trimeric complex, suggesting the positions of two domains of potential structural and functional homology, of one membrane-spanning alpha-helix approximately perpendicular to the membrane plane and of several tightly bound lipid molecules.

Chlorophyll↗
PubMed1988

Observations of restricted beam-induced specimen motion with small-spot illumination.

When lattice images of monolamellar paraffin crystals are recorded with a beam of diameter on the order of 1000 A, image contrast in part of the image is found to be at least three times higher than contrast in images obtained with conventional, flood illumination. Variations in contrast within these images, observed in previous experiments, are now found to be the result of radiation damage caused by the beam, which causes the crystal lattice to expand. This residual mode of beam-induced specimen motion, as well as distortions which propagate to adjacent, unirradiated areas of the specimen, are detected in a number of different ways. It thus appears that beam-induced specimen motion can be virtually eliminated over at least part of the illuminated area. Deleterious effects of the remaining mode of motion can be minimized by appropriate choice of exposure, beam-spot diameter, and spacing between exposed spots.

Microscopy, Electron↗
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

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↗
PubMed1984

Electron crystallographic studies of DNA.

The conformation of DNA in thin, platelet crystals is being studied by electron microscopy and diffraction, and by other techniques including circular dichroism spectroscopy. Specimens are prepared in the frozen-hydrated state for the electron microscope. Electron diffraction patterns from the crystals extend to 3.5 A, and lattice images with resolution to 6.5 A have been obtained. Electron diffraction patterns from fibers in the same preparations show a stacking distance between base pairs which is larger than in most DNA conformations. The data so far available can be interpreted to indicate that the DNA in the crystals is in a conformation which has not previously been reported and which is in the B family, unwound to have 12 base pairs per turn. Model calculations have been performed which support this interpretation and which show that a three-dimensional reconstruction, with even very limited data, could confirm the helix parameters.

Animals↗
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↗
PubMed1980

Electron diffraction from single crystals of DNA.

Crystals of DNA have been grown in a form suitable for study by electron diffraction and electron microscopy. Preliminary electron diffraction patterns of any type that have been obtained from single crystals of highly polymerized DNA. The patterns, obtained from frozen, hydrated crystals with the beam approximately parallel to the DNA strand axis, show a hexagonal geometrical arrangement with a (1,0) Bragg spacing of 23.1 A.

Animals↗