Simulation of transmission electron microscope images of biological specimens.

We present a new approach to simulate electron cryo-microscope images of biological specimens. The framework for simulation consists of two parts; the first is a phantom generator that generates a model of a specimen suitable for simulation, the second is a transmission electron microscope simulator. The phantom generator calculates the scattering potential of an atomic structure in aqueous buffer and allows the user to define the distribution of molecules in the simulated image. The simulator includes a well defined electron-specimen interaction model based on the scalar Schrödinger equation, the contrast transfer function for optics, and a noise model that includes shot noise as well as detector noise including detector blurring. To enable optimal performance, the simulation framework also includes a calibration protocol for setting simulation parameters. To test the accuracy of the new framework for simulation, we compare simulated images to experimental images recorded of the Tobacco Mosaic Virus (TMV) in vitreous ice. The simulated and experimental images show good agreement with respect to contrast variations depending on dose and defocus. Furthermore, random fluctuations present in experimental and simulated images exhibit similar statistical properties. The simulator has been designed to provide a platform for development of new instrumentation and image processing procedures in single particle electron microscopy, two-dimensional crystallography and electron tomography with well documented protocols and an open source code into which new improvements and extensions are easily incorporated.

Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm.

Using electron tomography, we have analyzed whether the Balbiani ring (BR) pre-mRNP particles in transit from the gene to the nuclear pore complex (NPC) are bound to any structure that could impair free diffusion through the nucleoplasm. We show that one-third of the BR particles are in contact with thin connecting fibers (CFs), which in some cases merge into large fibrogranular clusters. The CFs have a specific protein composition different from that of BR particles, as shown by immuno-EM. Moreover, we have identified hrp65 as one of the protein components of the CFs. The sequencing of hrp65 cDNA reveals similarities with hnRNP proteins and splicing factors. However, hrp65 is likely to have a different function because it does not bind to nascent pre-mRNA and is not part of the pre-mRNP itself. Taken together, our observations indicate that pre-mRNPs are not always freely diffusible in the nucleoplasm but interact with fibers of specific structure and composition, which implies that some of the posttranscriptional events that the pre-mRNPs undergo before reaching the NPC occur in a bound state.

Visualization of a large conformation change of ribosomes in Escherichia coli cells starved for tryptophan or treated with kirromycin.

Computer-aided electron tomography has been used to visualize ribosomes in Escherichia coli cells treated with kirromycin. This antibiotic stops bacterial growth by blocking the release of EF-Tu. GDP from the ribosome after GTP cleavage. Ribosomes in the kirromycin-treated cells are very compact, with the two subunits in close contact with each other. This closed structure is different from the open structure with spatially separated subunits that characterizes ribosomes in tryptophan-starved cells, giving deficiency for tryptophanyl.tRNA. A comparison of ribosomes in exponentially growing bacteria suggests that most ribosomes in an undefined working mode are in the closed conformation.

Physiologically dependent appearance of a low density region that corresponds to the tunnel through the 50S part of the 70S ribosome.

Ribosomes have different conformations in cells that are starved for a required amino acid (giving aminoacyl.tRNA starvation), or treated with kirromycin (blocking EF-Tu.GDP release), or are in exponential growth. A tunnel spans the 50S ribosome from a location facing the 70S ribosomal intersubunit space to the back side of the subunit in Escherichia coli cells. Here we have analyzed the internal low density region that corresponds to this tunnel in ribosomes in vivo. The data suggest that the tunnel is opened in connection with spatial separation of the subunits in ribosomes that have an empty A-site due to starvation for aminoacyl.tRNA. A region that corresponds to this tunnel can be found in the more compact structure of ribosomes in kirromycin-treated cells only after a substantial removal of low density material. This region is even less prominent in ribosomes in undefined working modes in growing bacteria. The data suggest that appearance of the tunnel through the 50S ribosomal subunit is working-mode dependent and it is not a characteristic feature of the major fraction of the ribosomal population in growing cells.

Automated correlation and averaging of three-dimensional reconstructions obtained by electron tomography.

We have developed a least-squares refinement procedure that in an automated way performs three-dimensional alignment and averaging of objects from multiple reconstructions. The computer implementation aligns the three-dimensional structures by a two-step procedure that maximizes the density overlap for all objects. First, an initial average density is built by successive incorporation of individual objects, after a global search for their optimal three-dimensional orientations. Second, the initial average is subsequently refined by excluding individual objects one at a time, realigning them with the reduced average containing all other objects and including them into the average again. The refinement is repeated until no further change of the average occurs. The resulting average model is therefore minimally biased by the order in which the individual reconstructions are incorporated into the average. The performance of the procedure was tested using a synthetic data set of randomly oriented objects with Poisson-distributed noise added. The program managed well to align and average the objects at the signal/noise ratio 1.0. The increase in signal/noise ratio was in all investigated cases almost equal to the expected square root of the number of objects. The program was also successfully tested on a set of authentic three-dimensional reconstructions from an in situ specimen containing Escherichia coli 70S ribosomes, where the immediate environment of the reconstructed objects may also contain variable amounts of other structures.

Maximum-entropy three-dimensional reconstruction with deconvolution of the contrast transfer function: a test application with adenovirus.

We have developed an objective, quantitative, and general algorithm to improve the fidelity of three-dimensional reconstructions made from electron micrographs while at the same time filtering much of the noise present in the recorded data. The new technique is called constrained maximum entropy tomography (COMET). The essence of the method is that it will produce the most featureless reconstruction that fits the projection data within their observational accuracy. In particular, the COMET procedure will minimise the detrimental effects of errors in the measured data and deconvolute the effects of the contrast transfer function. An objective test has been performed using COMET on a conventional image reconstruction obtained from cryo-electron micrographs of adenovirus. The density for hexon, the major coat protein of the virus, which is known to high resolution from X-ray crystallography, provided a known high-resolution control. The COMET reconstruction is in considerably better agreement with the crystallographic electron density than the original reconstruction, throughout the entire resolution range.

Starvation in vivo for aminoacyl-tRNA increases the spatial separation between the two ribosomal subunits.

Structures in situ of individual ribosomes in E. coli have been determined by computer-aided electron microscope tomography using a tilt series of positively stained embedded cellular sections. Amino acid starvation of a bacterial culture, causing a deficiency for aminoacyl-tRNA, induces a spatial separation between the ribosomal subunits compared with ribosomes in exponentially growing cells. Eight ribosomes from each growth condition were aligned to each other, and the two average structures were determined. Comparison of these suggests that the distance between the two subunits increases by approximately 3 nm upon starvation for aminoacyl-tRNA during protein synthesis. Ribosomes in most other states of the translational elongation cycle in exponentially growing cells show a more compact structure than previously realized.

Three-dimensional reconstruction of bovine intradural spinal root myelin by electron microscope tomography.

Electron microscope tomography was used to reconstruct three-dimensionally the configuration of heavy metal staining in bovine intradural spinal root myelin. Samples were fixed with glutaraldehyde, exposed to osmium tetroxide, embedded, thin sectioned, and finally stained with uranyl-acetate and lead citrate. Reconstructions up to 4.2 nm resolution showed a non-uniform distribution of stain in the planes of individual cytoplasmic appositions (major dense lines). In each reconstructed major dense line the stain was distributed in striated, well-defined structures. Those structures appear to be nearly parallel between neighboring major dense lines. The distribution of stain in the Schmidt-Lanterman cleft did not resemble the distribution of stain in the major dense line; however, weak striations were present. Evidence that the striated structures are not an artifact due to image calculation is discussed.

Spatial visualization of the maturing HIV-1 core and its linkage to the envelope.

The core of late states of maturing human immunodeficiency virus type 1 (HIV-1) has been visualized in three dimensions at approximately 7 nm resolution by electron microscopic tomography. After budding, approximately 25 nm thick precursor material is observed densely assembled inside the viral envelope. Upon proteolysis the core material is transported and condensed in the center of the virion. The core, 100 nm in length, spans the entire diameter of the virion showing a 40-60 nm wide free end and a narrow end approximately 20 nm. A model of the core is derived consisting of two fibers packed into a bilateral, elongated structure. Two ends of the fibers are compacted together, forming one narrow end of the core, while the two other fiber ends are situated more loosely together allowing for flexibility. Structural maturation of the virus could be reflected by the degree of compactness of the core. The narrow end of the core is observed attached to the envelope with a conspicuous core-envelope link (CEL).

Preparative displacement electrophoresis (isotachophoresis) of proteins on cellulose columns.

This paper describes the separation of proteins by displacement electrophoresis on columns packed with cellulose powder as a stabilizing medium. Cellulose has virtually no molecular sieving properties and thus differs from dextran, polyacrylamide, and agarose in this respect. Therefore, without the risk of unstacking, columns packed with cellulose permit conventional elution of the protein zones and the use of a counter flow (to increase the effective length of the bed). For the same reason, electroosmotic flow is less disturbing. A continuous elution-migration technique adapted to suit the special requirements of displacement electrophoresis gave better separation than was obtainable by conventional elution. Normal human serum and a fresh hemolysate from human erythrocytes were used as samples. An expression for the volume velocity of the boundaries is derived. This parameter can be used to determine the maximum duration of a run and a suitable pump speed when continuous elution or a counter flow is employed. The special advantages of displacement electrophoresis in cellulose beds are discussed as well as general disadvantages of the displacement technique, including the risk that proteins precipitate during a run.

The EDTA-treated expanded satellite tobacco necrosis virus: biochemical properties and crystallization.

The calcium ions normally present in the protein shell of the satellite tobacco necrosis virus (STNV) have been removed by EDTA. The radius of the EDTA-treated virion was found to be dependent upon pH. The radial increase was 2% at pH 5.0,4.5% at pH 6.5, and 7% at pH 7.0 as determined by analytical ultracentrifugation and X-ray crystallography. The virion could be recontracted by lowering the pH or by the addition of divalent cations, calcium ions being the most efficient. At pH 7.0 and above, the EDTA-treated virion was sensitive to ribonucleases and to trypsin. The cleavage site for trypsin is at Arg 28, which is near the N terminus of the shell domain of the subunit, but at a point which is well buried in the native structure. Crystals of the expanded STNV particles at pH 5.0 and 6.5 have been produced.

Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis).

The use of displacement electrophoresis (synonymous to isotachophoresis, steady-state stacking, and moving boundary electrophoresis) for recovery of DNA fragments from agarose and polyacrylamide gels is described. Complete recovery of DNA molecules ranging from oligonucleotides to 20 000-basepairs-long fragments was achieved. The DNA is recovered in a small volume (0.1-0.3 ml) and can be used directly in enzyme-mediated cleavage and ligation reactions. The recovered DNA contained no inhibitory contaminants as revealed by ligation or restriction enzyme cleavage.

Precolumn concentration of protein samples by displacement electrophoresis (isotachophoresis) followed by high-performance molecular sieve chromatography on a packed agarose column.

The use of displacement electrophoresis for the concentration of dilute protein solutions and the construction of a column suitable for this purpose are described. The concentrated protein zone can be pumped directly from the electrophoresis column into a gel-filtration column, which greatly reduces losses of protein. Recoveries of 95% or better were obtained even for small amounts of protein. The electrophoretically concentrated samples gave virtually the same elution profiles as did samples injected in a small volume without the use of electrophoretic preconcentration.

Molecular weight of beef heart aconitase and stoichiometry of the components of its iron-sulfur cluster.

Existing estimates of the molar content of iron and labile sulfide in aconitase are varying and deviate from integral numbers. The proposed model of the iron-sulfur cluster of inactive aconitase, suggesting it to contain a single [3Fe-4S] cluster, has prompted us to reinvestigate the basic physicochemical data of the enzyme to arrive at a more precise figure of the stoichiometry of Fe and S2-. The molecular weight of aconitase estimated from low speed sedimentation equilibrium was 80,900 +/- 2,200. Gel chromatography in 6 M guanidine HCl showed the presence of a single peptide chain of 710 residues, corresponding to a Mr of 78,400, while gel electrophoresis in presence of sodium dodecyl sulfate gave a value of 83,000. Both values are in reasonable agreement with the value obtained from sedimentation equilibrium. Protein determination by amino acid analyses, together with iron and sulfur analyses of 20 different preparations of greater than or equal to 95% purity, gives values of 2.9 +/- 0.2 Fe/mol and 3.9 +/- 0.2 S2-/mol. The data obtained are thus in agreement with the [3Fe-4S] model of the iron-sulfur cluster of inactive aconitase.

Recovery of proteins on a milligram scale from polyacrylamide electrophoresis gels, exemplified by purification of a retinol-binding protein.

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.