Heavy Metal Shadowing Research Articles

Structure of birnavirus-like particles determined by combined electron cryomicroscopy and X-ray crystallography

Birnaviruses possess a capsid with a single protein layer in contrast to most double-stranded RNA viruses infecting multicellular eukaryotes. Using freeze-drying and heavy metal shadowing, the capsids of two birnaviruses, infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus, as well as of an IBDV virus-like particle (VLP) are shown to follow the same T=13 laevo icosahedral geometry. The structure of the VLP was determined at a resolution of approximately 15 A (1.5 nm) by a combination of electron cryomicroscopy and a recently developed three-dimensional reconstruction method, where the scattering density is expressed in terms of symmetry-adapted functions. This reconstruction methodology is well adapted to the icosahedral symmetry of viruses and only requires a small number of images to analyse. The atomic model of the external capsid protein, VP2, recently determined by X-ray crystallography, fits well into the VLP reconstruction and occupies all the electron densities present in the map. Thus, similarly to the IBDV virion, only VP2 forms the icosahedral layer of the VLP. The other components of both VLP and IBDV particles that play a crucial role in the capsid assembly, VP1, VP3 and the peptides arising from the processing of pVP2, do not follow the icosahedral symmetry, allowing them to be involved in other processes such as RNA packaging.

The Surface Structure of Biomolecules Using Correlative Microscopy: TEM, SEM, STM and AFM

Freeze-drying followed by heavy metal shadowing is a long established and straight-forward TEM-approach to routinely study the structure of dehydrated macromolecular structures. Very thin specimens, such as isolated membranes or single macromolecules, can directly be adsorbed on Carbon-coated support grids.To attain the highest possible resolution, efforts were made to improve dehydration, metal shadowing and to minimize post-deposition changes in the metal film. Using our MIDILAB high vacuum cryotransfer system the freeze-dried and shadowed specimens are transferred into the microscope under high vacuum and cryo conditions. At no stage are the specimens exposed to air or warmed to ambient temperature. As a consequence, the stabilizing carbon film used in the conventional shadowing approach can be omitted and thereby images containing higher resolution structural features are recorded.

Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions.

The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.

Recent Progress In High-Resolution Shadowing For Biological Transmission Electron Microscopy

Freeze-drying followed by heavy metal shadowing is a long established and straight forward approach to routinely study the structure of dehydrated macromolecules. Very thin specimens such as isolated membranes or single macromolecules are directly adsorbed on C-coated grids. After rapid freezing the grids are transferred into a suitable vacuum equipment for freeze-drying and heavy metal shadowing.To improve the resolution power of shadowing films we introduced shadowing at very low specimen temperature (−250°C). To routinely do that without the danger of contamination we developed in collaboration with Balzers an UHV (p≤10-9 mbar) machine (BAF500K, Fig.2). It should be mentioned here that at −250°C the specimen surface acts as effective cryopump for practically all impinging residual gas molecules from the residual gas atmosphere.Common high resolution shadowing films (Pt/C, Ta/W) have to be protected from alterations due to air contact by a relatively thick C-backing layer, when transferred via atmospheric conditions into the TEM. Such an additional C-coat contributes disturbingly to the contrast at high resolution.

Electron microscopy of subnanometer surface features on metal-decorated protein crystals

Crystals of heavy riboflavin synthase from Bacillus subtilis were freeze-etched and vacuumcoated at normal incidence with 0.1 to 0.4 nm of gold and silver, respectively. This decoration technique was applied to probe the protein surface for preferential nucleation sites. Image processing of the electron micrographs revealed two particular decoration sites for silver and a different one for gold. According to X-ray crystallography, the riboflavin synthase molecules are spherical and smooth except for a surface corrugation of less than 1 nm, which can not be depicted by heavy-metal shadowing. Thus the decoration sites represent sites of specific physical-chemical interactions between the condensing metal and the protein. The decoration pattern correctly reflects the icosahedral symmetry of the almost spherical protein molecules. Owing to the molecule's symmetry, the position of these topochemical sites with respect to the symmetry axes can be localized within 5 Å. The packing of the molecules in the crystal can be directly observed on shadowed replicas. Only decoration, however, makes it possible to observe the exact orientation of the molecules within the crystal planes and to derive the true lattice constant along the 6-fold screw axis. This proves decoration to be a technique suitable for studying crystal packing and the molecular symmetry of protein complexes at high resolution. The technique can be applied to crystals that are not large enough or insufficiently ordered for X-ray crystallography.

T4 endonuclease V promotes the formation of multimeric DNA structures

Electron microscopy of UV-irradiated circular DNA molecules which had been treated with T4 endonuclease V revealed the formation of multimeric DNA structures in addition to the expected conversion of the superhelical DNA molecules into nicked circular and linear forms. The multimeric DNA molecules could be distinguished in electron micrographs from catenated molecules which were present in the original DNA preparation by a combination of rotary and single angle heavy metal shadowing. The complexity and frequency of these structures increased with time of reaction with endonuclease V. Their formation, as well as the endonuclease activity of enzyme, was dependent on UV irradiation of the DNA, and the complexes could be disrupted by prior phenol extraction and ethanol precipitation. Preparations of endonuclease V estimated to be 98% pure by mass promoted the same complex formation between DNA molecule as did preparations estimated to be only 5–10% pure. In addition to these intermolecular structures, the formation of complexes between regions on the same DNA molecules was manifest as discrete double-stranded ‘loops’ 200–300 base pairs in length. DNA ‘bubble structures’ were also observed and may represent folding of the ‘loops’ onto adjacent segments of DNA. These results suggest that at least one active form of T4 endonuclease V may be a multimetric complex of enzyme molecules in association with DNA.

A novel method for preventing collapse of air-dried specimens for heavy metal shadowing

Heavy metal shadowing is one of the oldest and most useful methods of enhancing the contrast of biological molecules for visualization by electron microscopy. A critical step in this method is the dehydration of the specimen prior to metal shadowing without causing distortion and collapse of the structure and consequent loss of the three-dimensional information. Two methods commonly employed today are glycerol spraying/vaccuum evaporation and freeze-drying. While glycerol spraying is the easier method of the two, it does not significantly prevent collapse artifact during air-drying and thus is limited to single extended molecules. Freeze-drying minimizes the collapse artifact of flattening and distortion due to surface tension. Freeze-drying preparations suffer their own problems of thermal vibrations and possible contamination by a eutectic of remaining salts and proteins.

The influence of the surface relief of thin sections of embedded, unstained biological material on image quality

Sections of resin embedded, aldehyde fixed T4 bacteriophages, adsorbed on bacterial envelopes, are observed unstained with different imaging modes and their surface reliefs analysed by heavy metal shadowing. It is found that details seen in bright-field and dark-field imaging reflect strongly the features of the relief. Most biological materials situated in the interior of the slice are not visible in bright-field; illustrating the few exceptions, the compact DNA of a bacterial virus that spans the entire section is visible by virtue of its density difference. By using resins with a chemically incorporated heavy metal the contrast is increased, as is the visibility of nearest neighbour distortions of the resin around the biological structures. The distortions studied with such a resin easily explain the blur observed with dark-field imaging in STEM of unstained sections with normal resin. It is confirmed that this blurring effect is eliminated either by ratio-contrast imaging that is less sensitive to thickness variations than the conventional modes or, obviously, by the abundant heavy metal stain commonly used. We then compared the experimental results with contrast calculations made previously and obtained excellent agreement between theory and experiment.

High resolution shadowing with pure carbon

Two test specimens, isolated purple membrane sheets of Halobacterium halobium and HPI layer of Deinococcus radiodurans, were used to investigate the condensation behaviour of pure carbon evaporated onto freeze-dried biological material. Carbon was evaporated at 15°, 45° and 90° elevation angle with, or without, rotating the specimen. On the exoplasmatic surface of the purple membrane a relief structure was found, causing a condensation pattern which correlates with the position of the bacteriorhodopsin molecules in a similar way as the decoration sites shown by Studer et al. [J. Cell Biol. 90 (1981) 153] after tantalum/tungsten shadowing. The very detailed relief structure of the HPI layer is also well portrayed by shadowing with pure carbon; the results are comparable to heavy metal shadowings shown by Wildhaber et al. [Ultramicroscopy 16 (1985) 411]. C-shadowing is a method complementary to heavy metal shadowing: Carbon condenses more or less amorphously. Because the deposition of carbon is enhanced in holes and depressions due to partial reflection effects, the resulting images resemble in some respect those from negatively stained preparation. The contrast of C-shadowed specimens is sufficiently high; no low-dose techniques have to be used.

Unshadowed myosin molecules: STEM mass-maps of myosin heads.

Myosin molecules were directly visualized without heavy metal shadowing by scanning transmission electron microscopy (STEM) under low dose conditions. The general appearance and dimensions of heavy metal-free molecules were similar to those of shadowed myosin, either after freeze-drying without or air-drying with glycerol. Two characteristic configurations of myosin head regions were found, a first type showing two pear-shaped heads with narrow necks and a second type showing two heads connected by an extra mass in the central regulatory domain where the light chains are located. The mass of the latter type (mol. wt. = 265 +/- 39 kd) is in excellent accordance with biochemical data whereas the mass of the first type is somewhat lower (mol. wt. 219 +/- 44 kd).

The structure of the photoreceptor unit of Rhodopseudomonas viridis

The thylakoid membrane of Rhodopseudomonas viridis contains extensive, regular arrays of photoreceptor complexes arranged on a hexagonal lattice with a repeat distance of 130 A. Single membrane sheets were obtained by mild treatment of the thylakoid fraction with the detergent Triton X-100. Heavy metal shadowing and electron microscopy of isolated thylakoids indicated a strong asymmetry of the membrane, showing a smooth plasmic and a rough exoplasmic side. Fourier processing of rotary-shadowed specimens showed the different surface relief on both sides of the membrane. Structural units on both sides were roughly circular and showed 6-fold symmetry at a resolution close to 20 A. The structural unit was characterised by a central core that seemed to extend through the membrane, protruding on the exoplasmic side. The core was surrounded by a ring showing 12 subunits on the plasmic side. Rotary-shadowed as well as negatively-stained membranes indicated a handedness of the structure. Treatment of thylakoid vesicles with higher detergent concentrations yielded a fraction of particles showing the same features as Fourier maps of the structural units. The isolated particles therefore appeared to represent structurally intact units of photosynthesis.

Electron microscopy of network structures in thermally-induced globular protein gels.

Thin sections of heat-set proteins gels formed from bovine serum albumin, insulin, lysozyme, ribonuclease, and alpha-chymotrypsin, have been studied by transmission electron microscopy. Micrographs have been interpreted as showing protein networks with strands between one and two times as thick as the native protein diameters. Considerable differences in the persistence characteristics, and frequencies of cross-linking, of the strands are observed, and there are variations in network homogeneity over long distances which correlate well with changes in gel opacity caused by alterations in pH and ionic strength. Evidence that artefacts are unlikely to have influenced these interpretations has been obtained in the BSA case in particular, by studying the aggregation process in solution, using alternative microscope approaches such as heavy-metal shadowing and negative staining. assuming that artefacts are absent, gel section micrographs have been simulated by a computer procedure, and the results suggest that, in most cases, the simplest interpretation of the microscope data is in terms of a "string of beads" model for the aggregation process, involving only moderately unfolded, and still globular, protein molecules. Other structural interpretations cannot be ruled out, however, as the degree of protein unfolding, and the exact mode of incorporation of the monomers into the network filaments, cannot be established by the microscope technique alone.

Helical crystals of sickle cell hemoglobin

Thin ribbon-like crystals are intermediates in the formation of large crystals of deoxyhemoglobin S from many individual fibers. The thin crystals show foldedover regions when observed by electron microscopy. Some crystals are sufficiently long to have several folds each separated by a distance of about 4.4 μm, suggesting that the crystals are helical in solution. The thickness of the crystals varies from 500 to 900 Å as shown by heavy-metal shadowing and by measurements of the thickness at the crossover point where an edge-on view of the crystal is obtained.

A New Freeze‐Etch Unit For Freeze‐Etch Rotary Shadowing, Low Temperature Freeze‐Fracturing and Conventional Freeze‐Etching

SUMMARYA new freeze‐etch unit is described in which one can make replicas where hydrated, frozen, fractured specimens rotate during specimen heavy metal shadowing. The freeze‐etch unit can also be used to do low temperature freeze‐fracturing and conventional freeze‐etching.

On the asymmetry of frog retinal rod outer segment disk membranes

Freeze-fracture studies on intact frog rod outer segments have been carried out using a device designed to minimize surface contamination and inadvertent specimen etching. When fractured and shadowed at −170°C, each double-membrane disk gives rise to two membrane fracture faces. In replicas with particularly favorable directions of heavy metal deposition, it is possible to infer the axial spacings between the membrane fracture faces by analyzing the widths of the oblique fracture jumps. The distance of each membrane fracture plane from the center of the disk can then be determined from these axial spacings and the structural centrosymmetry of the disk. Our analysis indicates that each membrane fracture plane is located at a distance of ∼47 Å from the coordinate origin at the center of the disk. This coordinate corresponds in position to the electron density minimum of the membrane. Our results therefore support the original hypothesis by Clark and Branton (1968) that disk membranes fracture along some interior hydrophobic plane, presumably the region of terminal methyl groups at the center of the lipid bilayer. Fracture face A is characterized by a rough, particulate appearance, and represents the hydrophobic face of that half of the disk membrane adjacent to the cytoplasmic matrix. Face B is smooth, and represents the hydrophobic face of that half of the membrane adjacent to the intradisk compartment. When these membrane fracture faces are compared with the characteristic textures observed in pure lipid bilayers and in rhodopsin-lipid recombinant membranes, it appears that in frozen disk membranes rhodopsin is restricted to the cytoplasmic half of the membrane. The smooth B face has the appearance of a lipid monolayer. Although the A face particles have a rather low surface relief, it is still possible to estimate their apparent size and shape. These particles are roughly circular; the range of diameters is about 120–140 Å, when measured in the projected direction of heavy metal shadowing. Optical diffraction patterns of the A face show broad intensity bands centered at ± ( ∼ 1 130 A ̊ ). When examining the published equatorial X-ray diffraction data from frog outer segments, it is apparent that there is no broad band of intensity within the interval ( 1 150 A ̊ ) to ( 1 70 A ̊ ), corresponding to the optical diffraction data. Accordingly, it must be postulated that at physiological temperatures, the distribution of matter within the plane of the disk membrane does not correspond to the distribution evident in the A fracture face. Hence, the A face particles appear to represent artifactual, derivative phenomena. An explanation in terms of lateral phase separations of disk membrane components is developed. The consequences of such an hypothesis for the etching behavior of the B face are derived in the context of those parameters which are known to affect the passage of water molecules across lipid monolayers.

Further electron microscope studies of the conidium of Alternaria brassicicola.

The freeze-etching technique, aldehyde fixation and heavy metal shadowing of wall material were used in an electron microscope study of the maturing spores of Alternaria brassicicola (Schw.) Wiltshire. The walls are composed of fibres, probably of chitin. The plasmalemma has rectangular grooves in its outer surface and corresponding ridges on the inner one; both surfaces bear particles of two distinct sizes. Endoplasmic reticulum may be lamellated or vesicular and its involvement in wall formation is confirmed; vesicles produced by the endoplasmic reticulum fuse with the plasmalemma. The structure of nuclei, mitochondria and vacuoles is also demonstrated.

Correlation of structure with function in Spinacea oleracea chloroplasts

Spinach chloroplasts, isolated in isotonic sucrose medium, were sonically ruptured and separated by differential centrifugation into three main fractions: a green precipitate, a colourless supernatant and a yellow, low density lipid-containing layer. These fractions were analyzed for their physical, chemical and biochemical properties. Comparison of a thin section of an isolated osmium-stained chloroplast with a thin section of osmium-stained green precipitate, by electron microscopy, shows that the particles in the green precipitate correspond both in thickness and lateral dimensions to the lamellar structure of the isolated chloroplast. Heavy metal shadowing of both air-dried and lyophilized green precipitate demonstrates the presence of lamellar structures from 10,000 to 20,000 A in diameter. The lamellar structure, totaling 160 A in thickness, is composed of two layers; each layer is made up of granular subunits 100 A thick. Chlorophyll to nitrogen weight ratios are fairly constant for green lamellar structures from 800 to 20,000 A in diameter, indicating that chlorophyll is uniformly distributed throughout the lamellar structure of the chloroplast. The similarity of relative photosynthetic pigment concentrations over the visible region, of the Hill reaction activity and of the carbon dioxide fixation capacities for the various sized particles in the green precipitate suggests that the smallest lamellar fragments used in these experiments are large in comparison with the smallest fragment which converts electromagnetic to chemical energy. A model for chloroplast lamellar structure is proposed on the basis of the results obtained. The supernatant, by electron microscopy, is seen to consist principally of oblately spherical particles, 100 A thick and 200 A in diameter. These particles are indistinguishable from the main water-soluble protein of the chloroplast with sedimentation coefficient 16 (Fraction I protein). About 90% of the total carboxydismutase activity is associated with the supernatant proteins. In addition, these proteins are able to convert fructose-6-phosphate into other photosynthetic carbon cycle intermediates. This supernatant must be added to the green precipitate in order to obtain maximum carbon dioxide fixation rates. Electron micrographs of unfractionated osmium-fixed chloroplast sonicate show large osmium-stained spherical objects corresponding in size and staining properties to the osmiophyllic granules observed in thin sections of osmiumstained chloroplast in the intact leaf. Similar granules, 500 to 1500 A in diameter, were obtained when the yellow, low density lipid layer was fixed with osmium and then shadowed.