Mass determination of the unit cell of the innermost chorionic layer in Drosophilidae by scanning transmission electron microscopy.

The innermost chorionic layer (ICL) in eggshells of Drosophila melanogaster is a naturally occurring patchwork of thin three-dimensional crystalline plates located between the inner endochorion and the vitelline envelope. The mass-per-unit area of the ICL has been measured from scanning transmission electron microscope images of isolated unstained material and it was possible to distinguish up to four layers with the majority of the crystalline sheets being one to three layers thick. Taking into account the unit cell areas for the different crystals, we have estimated the mean ICL subunit sizes to be 36 kDa for Drosophila melanogaster, 35 kDa for Drosophila auraria, and 33 kDa for Drosophila teissieri. The results suggest that the three different Drosophilidae species have very similar average subunit masses.

Structural studies of the surface projections of Chlamydia trachomatis by electron microscopy.

Rod-like projections on the surface of Chlamydia trachomatis have been studied by a combination of computer image analysis and electron microscopy. The rods, c. 60-80 A in diameter and c. 500 A in length, were found on the surface of prokaryocells of C. trachomatis inserted in the cytoplasmic membrane through a ring-like structure in the outer membrane. The rod-like structures were found at all stages of the life cycle, even in very small elementary bodies (EBs) of C. trachomatis and in vesicles < 0.2 micron. Computer image analysis of isolated rods indicated that they comprise helically arranged subunits with a periodicity of c. 50 A. From their localisation and distribution, these structures may be related to the proliferation, or to the infectivity, of chlamydiae.

Structure of the Lethocerus troponin-tropomyosin complex as determined by electron microscopy.

Native troponin-tropomyosin complex was isolated from Lethocerus indicus indirect flight muscle and tested for function. It was shown by rotary shadowing and by forming paracrystals on monolayers that the regulatory complex consists of a troponin head region approximately 130 A in diameter and a 400-A-long troponin T-tropomyosin tail. The complex forms paracrystals at the air-water interface on a positively charged monolayer. The globular head packs in rows 380 A apart which are bridged by the tail domain. Filamentous paracrystals were obtained by adding Mg2+ ions to the troponin-tropomyosin sample. These showed globular domains arranged in a regular pattern along "ribbon"-like filaments. The spacing of the repeat was determined to be 380 A.

Three-dimensional structure of NADH-dehydrogenase from Neurospora crassa by electron microscopy and conical tilt reconstruction.

NADH-dehydrogenase (Complex I) is the first complex of the mitochondrial respiratory chain. It is an amphipatic molecule located in the inner mitochondrial membrane and is composed of at least 35 unique subunits encoded by both mitochondrial and nuclear DNA. The whole complex was isolated in detergent from the fungus Neurospora crassa. It is very stable in its isolated form and was analysed as such by electron microscopy. Its mass, determined by dark-field scanning electron microscopy was estimated as 1.12 MDa. The complex was imaged by transmission electron microscopy, by negative staining and by cryo-electron microscopy. A three-dimensional model, with a resolution estimated at 35 A, was calculated from images of negatively stained complexes by the random conical tilt reconstruction technique. This model confirms the general L-shape of the molecule, with arms of equal length and corroborates the hypothesis of a subdivision of the whole complex into three functional domains. Immuno-labelling of the 49 kDA subunit of the peripheral arm allowed its localization within the complex. This is a first step in the subunit mapping of Complex I and the understanding of its activity.

Improved immuno double labelling for cell and structural biology.

Colloidal gold is the most widely used electron dense marker in biological electron microscopy. The development of procedures for making gold particles of very defined sizes has made double or even multiple labelling possible using gold of two or more different sizes. Lately a new type of electron dense marker has been developed consisting of ligand-stabilized metal atom clusters rather than colloidal particles. The differences between these two types of markers are highlighted and the advantages of using metal atom clusters for immuno labelling of certain biological specimens are discussed. Possible methods of distinguishing two such cluster labels in double labelling experiments are reviewed.

Unconventional immuno double labelling by energy filtered transmission electron microscopy.

A new method of immuno double labelling of biological specimens with a very high spatial resolution is presented. The advantage over conventional techniques is the possibility of using two very small labels leading to higher labelling efficiency, better penetration into the specimen and reduced steric hindrance between labels at closely spaced sites. The two labels are distinguished by their electron energy loss spectra using principal component analysis and then identified by comparison with an external standard using discriminant function analysis. The method is tested on samples of insect flight muscle labelled with 8 nm colloidal gold and silver and the statistical reliability of the classification is assessed. Extensions of the method are suggested and its potential for biological research is discussed.

Cytoskeletal proteins of insect muscle: location of zeelins in Lethocerus flight and leg muscle.

Asynchronous insect flight muscles produce oscillatory contractions and can contract at high frequency because they are activated by stretch as well as by Ca2+. Stretch activation depends on the high stiffness of the fibres and the regular structure of the filament lattice. Cytoskeletal proteins may be important in stabilising the lattice. Two proteins, zeelin 1 (35 kDa) and zeelin 2 (23 kDa), have been isolated from the cytoskeletal fraction of Lethocerus flight muscle. Both zeelins have multiple isoforms of the same molecular mass and different charge. Zeelin 1 forms micelles and zeelin 2 forms filaments when renatured in low ionic strength solutions. Filaments of zeelin 2 are ribbons 10 nm wide and 3 nm thick. The position of zeelins in fibres from Lethocerus flight and leg muscle was determined by immunofluorescence and immunoelectron microscopy. Zeelin 1 is found in flight and leg fibres and zeelin 2 only in flight fibres. In flight myofibrils, both zeelins are in discrete regions of the A-band in each half sarcomere. Zeelin 1 is across the whole A-band in leg myofibrils. Zeelins are not in the Z-disc, as was thought previously, but migrate to the Z-disc in glycerinated fibres. Zeelins are associated with thick filaments and analysis of oblique sections showed that zeelin 1 is closer to the filament shaft than zeelin 2. The antibody labelling pattern is consistent with zeelin molecules associated with myosin near the end of the rod region. Alternatively, the position of zeelins may be determined by other A-band proteins. There are about 2.0 to 2.5 moles of myosin per mole of each zeelin. The function of these cytoskeletal proteins may be to maintain the ordered structure of the thick filament.

Gold/Fab immuno electron microscopy localization of troponin H and troponin T in Lethocerus flight muscle.

The position of the large troponin complex relative to myosin crossbridges in Lethocerus flight muscle (IFM) has been probed by electron microscopy (EM) using monoclonal antibodies against troponin T (TnT) and troponin H (TnH), a heavy troponin component found in several insect muscles. Infiltration of gold-tagged and plain Fab fragments into glycerinated IFM before fixation established in non-overlap fibers that the beads every 38.7 nm along thin filaments are troponin. Original and optically filtered EM images from 25 nm longitudinal and 15 nm cross-sections of partially overlapped fibers suggests that epitopes on both TnT and TnH are very close to the rear crossbridge of the rigor double chevron. When Fab was infiltrated into relaxed fibers and ATP was subsequently removed, the resulting rigor crossbridge lattice was disrupted by antibody labeling of the troponin. The results confirm that the lattice of rigor crossbridges and troponin are congruent and suggest that crossbridges may interact with troponin in IFM, possibly serving as a partial basis for the stretch activation characteristic of this muscle.

Radial mass density functions of vitrified helical specimens determined by scanning transmission electron microscopy: their potential use as substitutes for equatorial data.

Using STEM dark field images, we have determined linear mass densities and radial density profiles of vitrified helical particles. The samples studied are: TMV, RNA-free helical polymers of TMV coat protein (TMV-P), Salmonella typhimurium bacterial flagellar filaments and Escherichia coli pili. The difference between the profiles obtained for TMV and TMV-P shows a maximum at a radius of about 4 nm, corresponding to the RNA in TMV. Of the peaks that are resolved in X-ray diffraction analysis we can resolve the ones for TMV at radii of approximately 4.2 and approximately 6.7 nm and a shoulder at approximately 7.8 nm. Density peaks in bacterial flagellar filaments appear at radii of approximately 4.2, approximately 6.5, approximately 8.5, and approximately 10.5 nm. Accurate mass data can be obtained if the filaments are embedded in ice layers of uniform thickness; their diameters need to be similar to that of the mass standard (TMV) when these data are measured in a comparative manner. Ice layers are often not uniform, and thickness variations are well revealed in STEM dark field. The signal-to-noise ratio and contrast for the transverse projections are lower than those measured for freeze-dried specimens: half an order and one order of magnitude, respectively. The thinnest uniformly thick ice layer still containing a single layer of particles is approximately 10-15 nm thicker than the particles. Radial mass density functions that are directly determined in STEM may have a potential use as substitutes for the unreliable equatorial data in helical reconstructions of TEM bright field images of vitrified specimens.

The aerolysin membrane channel is formed by heptamerization of the monomer.

The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS-PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two-dimensional membrane crystals. Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids. A noise-filtered image of the best crystalline sheets reveals a structure with 7-fold symmetry containing a central strongly stain-excluding ring that encircles a dark stain-filled channel 17 A in diameter. The ring is surrounded by seven arms each made up of two unequal sized domains. By combining projected views and side-views, a simplified model of the aerolysin channel complex has been constructed. The relevance of this structure to the mode of action of aerolysin is discussed.

A method for determining the periodicity of a troponin component in isolated insect flight muscle thin filaments by gold/Fab labelling.

Insect flight muscle has a large component (Tn-H) in the tropomyosin-troponin complex that is not present in vertebrate striated muscle thin filaments. Tn-H is shown by gold/Fab labelling to be present at regular intervals in insect flight muscle thin filaments. The Fab fragment of a monoclonal antibody to Tn-H was conjugated directly with colloidal gold and this probe used to label isolated thin filaments from the flight muscle of Lethocerus indicus (water bug). The distribution of gold particles seen in electron microscope images of negatively stained thin filaments was analysed to show that the probe bound to sites having a periodicity of approximately 40 nm, which is the expected value for the tropomyosin-troponin repeat. Conjugates of Fab with colloidal gold particles of 3 nm diameter labelled almost all sites. Conjugates with gold particles of 5 nm and 10 nm diameter labelled less efficiently (70% and 30%, respectively) but analysis of the distribution of inter-particle intervals among a number of filaments again gave the same fundamental spacing of 40 nm. The error in the measurements (standard deviation approximately +/- 4.2 for 5 nm gold/Fab) is less than earlier estimates for the size of the gold/Fab complex. Measurements on gold/Fab in negative stain suggest that the bound Fab contributes a shell about 2 nm in thickness around the gold particle. The radius of the probe (about 4.5 nm for 5 nm gold/Fab) would then be consistent with the value of error found. The size of the probe suggests that the gold particle binds to the side of the Fab molecule, rather close to the antibody combining site. The potential resolution of the technique may thus be better than originally expected.

Three-dimensional reconstruction of innermost chorion layer of Drosophila grimshawi and Drosophila melanogaster eggshell mutant fs(1)384.

A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.

Identification and electron microscopic analysis of a chaperonin oligomer from Neurospora crassa mitochondria.

A 7-fold symmetric particle has been identified in Neurospora crassa which is most probably the mitochondrial chaperonin. The particle, about 12 nm in diameter, appears in preparations of cytochrome reductase, and is shown to contain a 60 kd protein which cross-reacts with anti-GroEL antibodies. Results of STEM mass measurement suggest that the particle is composed of 14 subunits. A preliminary interpretation of the structure of the particle based on electron microscopy is given. Its quaternary structure and molecular weight are similar to those of the recently discovered family of particles called chaperonins, found in bacteria, chloroplasts and mitochondria.

Electron microscopic study of crystals of the Xenopus laevis transcription factor IIIA-5S ribosomal RNA complex.

A novel method has been developed to grow crystals of the Xenopus laevis transcription factor IIIA-5S RNA complex directly on grids for examination by electron microscopy. Microcrystals were examined in negative stain and in thin sections to reveal a hexagonal lattice with unit-cell dimensions a = b = 87.1 +/- 4.4 A and c = 143.8 +/- 12.7 A. Optical diffraction patterns from micrographs were obtained about the major crystal axes extending to about 40-A resolution. A packing scheme is proposed for which there are three or six transcription factor IIIA-5S RNA complexes in the unit cell related by 3(1) symmetry along the long cell axis. This would require that the 5S RNA molecules are arranged end-to-end, with the terminal loops of adjacent molecules overlapping.

A tunnel in the large ribosomal subunit revealed by three-dimensional image reconstruction.

A better understanding of the molecular mechanism of protein biosynthesis depends on the availability of a reliable model for the ribosome particle. The application of a diffraction technique, namely, three-dimensional image reconstruction from two-dimensional sheets of the large ribosomal subunits of Bacillus stearothermophilus at a resolution of 30 angstroms is described. The resulting three-dimensional model shows at least four projecting arms, arranged radially near the presumed interface with the 30S subunit. The projecting arms are positioned around a cleft, which turns into a tunnel with a length of 100 to 120 angstroms and a diameter of up to 25 angstroms. This tunnel spans the particle and may provide the path taken by the nascent polypeptide chain.

Methods for specimen thickness determination in electron microscopy. II. Changes in thickness with dose.

The electron diffraction patterns of tilted thin crystals were used to determine the unit cell size in the direction normal to the supporting film. The method revealed a considerable dose-dependent thinning or shrinkage. Using a variety of specimens and stains, we found that this amounted to a 50% reduction in volume and could be attributable to two causes. Firstly, the specimen is held to the supporting film so that volume changes can only occur through changes in thickness. Secondly, the decrease in volume is associated with a dose-induced mass loss which is greatly suppressed at liquid nitrogen temperatures.

Methods for specimen thickness determination in electron microscopy.

A new method is described for specimen thickness determination in transmission electron microscopy. This is carried out by marking specimens with gold particles and analysing the images of a tilt series by computer. The method makes it possible to distinguish populations of particles on different planes and calculate the distance between the planes with statistical variation. We have applied it to carbon films as test objects and compared the results with those obtained by transverse sectioning, STEM mass measurement, optical density and frequency change of a quartz crystal oscillator. We have then used the method for thickness measurement of multilayered protein crystals and thin sectioned cells.

Three-dimensional reconstruction of innermost chorion layer from Drosophila melanogaster.

A low-resolution three-dimensional structure of the crystalline innermost chorion layer (ICL) has been calculated from electron microscope images of tilted negatively stained crystals. The isolated ICL is a single layer, about 12 nm thick and appears to be made up of two types of subunits, each approximately 3 nm in diameter, arranged regularly as groups of four heterodimers in space group C222. Linking density between these groups of subunits, maintaining the integrity of the layer, appears to be confined mainly to the outer surfaces of the ICL.