Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.     

Correlation analysis of gap junction lattice images.

Fourier averages of connexon images computed from low-irradiation electron micrographs of isolated negatively stained gap junction domains exhibited differences in stain distribution and connexon orientation. To analyze these polymorphic structures, correlation averaging methods were applied to images from negatively stained and frozen-hydrated specimens. For the negatively stained specimens, separate averages over two subsets of connexons with differing degrees of stain accumulation in the axial channel were obtained. Two populations of connexons with opposite skew orientations were distinguishable within a single junctional domain of a frozen-hydrated specimen. Correlation maps calculated using the left- and right-skewed references showed that the selected connexons tend to locally cluster. Using correlation methods to analyze packing disorder in a typical connexon lattice, we estimated the root-mean-square variation in the nearest neighbor pair separation to be approximately 11% of the lattice constant. Displacements of the connexons relative to each other increased with increasing pair separation in the lattice, rather like a liquid, although long-range orientation order was conserved as in a crystal. These results support the hypothesis that the hexagonal ordering of the connexons results from short-range repulsive forces.     

A compact three-dimensional crystal form of the large ribosomal subunit from Bacillus stearothermophilus

A new form of well-ordered three-dimensional crystals of intact 50 S ribosomal subunits from Bacillus stearothermophilus have been obtained. Electron micrographs of positively stained sections of these crystals revealed that the ribosomal particles are packed closely. The cell parameters have been determined. Representative electron micrographs and their computed contoured filtered images are shown.     

Projected structure of the pore-forming OmpC protein from Escherichia coli outer membrane.

A single-projection structure analysis of a bacterial outer membrane protein, OmpC, has been carried out by electron microscopy of frozen hydrated specimens. Two distinct crystal polymorphs have been observed in the frozen-hydrated samples, and projection structures of both forms have been obtained to a resolution of 13.5 A. Preliminary examination of negatively stained samples revealed the expected, trimeric appearance of pores in the OmpC specimens. Electron microscopy of unstained, frozen-hydrated OmpC reveals the trimeric pore structure with equal clarity. In addition, the overall molecular envelope of the protein is readily discerned, and a major lipid-containing domain can also be seen. Because of the small coherent patch size, mosaic disorder, and unpredictable polymorphism of the presently available specimens, three-dimensional reconstruction of frozen-hydrated OmpC has not been carried out.     

On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.     

A survey of 50 S ribosomal subunits by dark field electron microscopy

A survey of dark field electron micrographs of the 50 S ribosomal subunit of E. coli has been performed and supplemented, for comparative purposes, by examination of negatively stained or metal shadowed specimens in the bright field mode. Attention was directed to the so-called "crown" and "kidney" views. The elongated appendage seen in negatively stained crown profiles was not observed in unstained or positively stained samples examined in dark field; these showed only symmetrical crown profiles regardless of changes in buffer type and drying method and of the presence or absence of uranyl acetate treatment and glutaraldehyde fixation. The crown view occasionally displayed a bifurcation in one of the lateral lobes, while the kidney profile showed a groove near the base of the convex edge. Uranyl acetate treatment produced delicate stripes which may give an indication of the surface RNA distribution.     

Two-dimensional crystalline sheets of Bacillus stearothermophilus 50S ribosomal particles.

Two-dimensional crystalline sheets of the large ribosomal subunit from Bacillus stearothermophilus have been obtained using a slightly modified procedure to that for growing three-dimensional crystals of the same material. The crystalline subunits are packed within monolayers in a relatively small unit cell, the dimensions of which are closely related to those observed for two forms of the three-dimensional crystals. The packing symmetry is p121, and the optical diffraction patterns of micrographs of negatively stained crystals extend to approximately 3.0 nm.     

Structural analysis of thermosome from hyperthermophilic archaeon Thermococcus

The purification and characterization of thermostable chaperonin of the thermosome family from hyperthermophilic archaeon Thermococcus profunds are described. The purified thermosome is a homooligomeric complex and an ATPase with maximal activity at 80 degrees C. The electron micrographs obtained from negatively stained as well as frozen-hydrated specimen showed an eight-fold symmetry of chaperonin. They were about 15 nm height and 16 nm in diameter with a central cavity of 5 nm. In order to understand the ATPase cycling of thermosome, we analyzed the oligomeric structure of thermosome treated with several nucleotides.     

Studies on the structure of animal ribosomes. VIII. Application of a digital image processing method to the enhancement of electron micrographs of small ribosomal subunits

Electron micrographs of negatively stained small subunits of rat liver ribosomes have been processed by computer-assisted accumulation. The resulting micrographs are characterized by a significant noise suppression and enhancement of image details.     

Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.     

Three-dimensional reconstruction and averaging of 30 S ribosomal subunits of Escherichia coli from electron micrographs

From the micrographs of a tilt series, several particles of negatively stained 30 S ribosomal subunits of Escherichia coli were three-dimensionally reconstructed. Three of them showing similar orientation with respect to the supporting foil were averaged after alignment by newly developed three-dimensional correlation methods. As a main result we found a stained channel-like structure inside the particle. We tentatively propose that this corresponds, at least partially, to positively stained segments of the 16 S RNA.     

Cryo-EM of the native structure of the calcium release channel/ryanodine receptor from sarcoplasmic reticulum.

The native structure of the calcium release channel (ryanodine receptor) from rabbit skeletal muscle has been analyzed in two dimensions from electron micrographs of frozen hydrated specimens. Within a resolution of 3.0 nm there is excellent agreement between the structure as seen in vitreous water and in negative stained specimens. Features seen in the three-dimensional reconstruction of the negatively stained channel can be identified in the projection of the unstained receptor.     

Effects of magnesium and formaldehyde on the sedimentation behavior of rat liver ribosomes

When rat liver ribosomes were dissociated by removing their magnesium, the two subunits had s,w of 52.6 and 28.1 S, and appeared to be quite asymmetrical. In 4M urea they became even more elongated. On restoration of magnesium, both subunits assumed more compact forms, 57.6 and 31.7 S, but only 28% of them formed larger aggregates. After brief treatment with formaldehyde, the magnesium-free subunits still elongated in urea, but to a lesser degree than the controls. When magnesium was restored they changed to 53.4 and 31.7 S forms, but did not, aggregate. The ribosomal subunits were also separated by reducing the bound magnesium to a low level, then adding 2.2M urea. After the urea had been dialyzed out, most of the large subunits were still in a compact form and when the magnesium concentration was increased, most of the subunits did not reassociate to whole ribosomes. Formaldehyde-treated subunits, however, did not reassociate. Even in their most compact forms, 60.0 and 35.5 S, both subunits had high frictional ratios. The 60.0 and 31.7 S forms were assumed to be the shapes that are seen in electron micrographs of negatively stained ribosomes. Both subunits seemed to be highly hydrated. Since the large subunit appeared to contain twice as much water as the small one, it might be a structure with either a deep channel or a hollow center.     

Three-dimensional structure and subunit topology of the V(1) ATPase from Manduca sexta midgut

The three-dimensional structure of the Manduca sexta midgut V(1) ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V(1) complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V(1) complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl(2)-induced disulfide formation. Treatment of V(1) with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V(1) decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl(2) was added to V(1) in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl(2) was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.     

A disulfide conjugate between anti-tetanus antibodies and HIV (37-72)Tat neutralizes tetanus toxin inside chromaffin cells

The structure of the V1 ATPase from the tobacco hornworm Manduca sexta has been determined from electron micrographs of isolated, negatively stained specimens. The resulting images clearly show a pseudohexagonal arrangement of six equal-sized protein densities, presumably representing the three copies each of subunits A and B, which comprise the headpiece of the enzyme. A seventh density could be observed either centrally or asymmetrically to the hexamer. The maximum diameter of the V1 complex in the hexagonal projection is 13 nm with each of the six peripheral densities being 3-4 nm in diameter.     

Helical arrays of Escherichia coli small ribosomal subunits produced in vitro.

In vitro conditions have been determined for obtaining ordered helical ribbons of small ribosomal subunits from Escherichia coli. These ribbons, suitable for study by three-dimensional reconstruction, are the first ordered arrays of ribosomes or ribosomal subunits to be produced in vitro.Although small ribosomal subunits remain in solution for extended periods (up to 6 months) during this procedure, their structural integrity, as assessed by acrylamide/agarose gel electrophoresis, by sucrose gradients, and by electron microscopy, is not significantly altered.Electron micrographs of ribbons of small subunits diffract to 60 Å resolution. Optical diffraction patterns suggest that adjacent subunits within helical ribbons are related by a 2-fold screw parallel to the long axis of the ribbon and the helical repeat distance measured from electron micrographs is 220 Å.     

Projected structure of unstained, frozen-hydrated T-layer of Bacillus brevis.

This paper presents the projected structure of the T-layer of Bacillus brevis, obtained from electron microscopic studies of the unstained protein layer in the frozen-hydrated state. Computer image processing is used to correct for the effects of the contrast transfer function, and to increase the signal-to-noise ratio by lattice averaging. The results obtained show a good agreement with those previously obtained using negatively stained specimens. It is shown that the contrast of T-layer embedded in ice can be approximated to pure phase contrast.     

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.     

Electron microscopy of the stacked disk aggregate of tobacco mosaic virus protein. II. The influence of electron irradiation of the stain distribution

Optical and computer analysis of bright field electron micrographs of the same negatively stained stacked disk specimens subjected to different electron doses shows that the stain migrates and redistributes itself over the protein surfaces during irradiation. The analysis suggests that the main factors responsible for this migration are the contraction of the stain inevitably brought about by the irradiation, and associated surface energy effects. The morphology of the protein has a strong influence on the path of the migration.A major consequence of these changes in stain distribution is that conventional bright field micrographs taken under normal dosage conditions tend to give a somewhat misleading representation of the specimen's original structure.Migration of the stain under the electron beam is likely to be a fairly general phenomenon and it therefore seems important to take bright field micrographs of negatively stained specimens using the smallest possible electron doses.     

Crystallization of 70 S ribosomes and 30 S ribosomal subunits from Thermus thermophilus

Well-ordered three-dimensional crystals of 70 S ribosomes and 30 S ribosomal subunits from extremely thermophilic bacteria Thermus thermophilus have been obtained. Positively stained thin sections of the crystals have been analyzed by electron microscopy. Redissolved crystalline ribosomes and small ribosomal subunits reveal sedimentation constants of 70 S and 30 S, respectively, and are functionally active in the poly(U)-system.