Molecular cryo-electron tomography of vitreous tissue sections: current challenges.

Electron tomography of vitreous tissue sections (tissue TOVIS) allows the study of the three-dimensional structure of molecular complexes in a near-native cellular context. Its usage is, however, limited by an unfortunate combination of noisy and incomplete data, by a technically demanding sample preparation procedure, and by a disposition for specimen degradation during data collection. Here we outline some major challenges as experienced from the application of TOVIS to human skin. We further consider a number of practical measures as well as theoretical approaches for its future development.

Resolving the geometry of biomolecules imaged by cryo electron tomography.

In this paper, we describe two methods for computerized analysis of cryo electron tomography reconstructions of biomolecules. Both methods aim at quantifying the degree of structural flexibility of macromolecules and eventually resolving the inner dynamics through automatized protocols. The first method performs a Brownian dynamics evolution of a simplified molecular model into a fictitious force field generated by the tomograms. This procedure enables us to dock the simplified model into the experimental profiles. The second uses a fuzzy framework to delineate the subparts of the proteins and subsequently determine their interdomain relations. The two methods are discussed and their complementarities highlighted with reference to the case of the immunoglobulin antibody. Both artificial maps, constructed from immunoglobulin G entries in the Protein Data Bank and real tomograms are analyzed. Robustness issues and agreement with previously reported measurements are discussed.

Anti-cooperativity in diffusion-controlled reactions with pairs of anisotropic domains: a model for the antigen-antibody encounter.

The encounter between anisotropic agents in diffusion-controlled reactions is a topic of very general relevance in chemistry and biology. Here we introduce a simplified model of encounter of an isotropic molecule with a pair of partially reacting agents and apply it to the encounter reaction between an antibody and its antigen. We reduce the problem to the solution of dual series relations, which can be solved iteratively, yielding the exact solution for the encounter rate constant at any desired order of accuracy. We quantify the encounter effectiveness by means of a simple indicator and show that the two binding centers systematically behave in an anti-cooperative fashion. However, we demonstrate that a reduction of the binding active sites allows the composite molecule to recover binding effectiveness, in spite of the overall reduction of the rate constant. In addition, we provide a simple formula that enables one to calculate the anti-cooperativity as a function of the size of the binding site for any values of the separation between the two active lobes and of the antigen size. Finally, some biological implications of our results are discussed.

Dynamics of antibodies from cryo-electron tomography.

The issue of protein dynamics and its implications in the biological function of proteins are arousing greater and greater interest in molecular biology. In cryo-electron tomography experiments one takes several snapshots of a given biological macromolecule. In principle, a large enough collection of snapshots may then be used to calculate its equilibrium configuration in terms of the experimentally accessible degrees of freedom, and hence estimate its potential energy. Consequently, one could analyze the biological functions of biomolecules by directly accessing their dynamics. In this work, we analyze the results of cryo-electron tomography experiments on monoclonal murine IgG2a antibodies. With the aid of a novel software for image processing, we measure the equilibrium distribution of the angles which describe the configuration of the molecule. This helps us shed some critical light on recent results from X-ray crystallography. We then build a model of the antibody dynamics, which enables us to use the measured angular distribution in order to derive an explicit expression of the IgG potential energy. Finally, as a preliminary application of our results, we investigate the dynamical effects in the rate of formation of the antigen-antibody encounter complex. In particular, we suggest that the dynamics of antibodies operates in the direction of decreasing anticooperativity of the two antigen binding arms.

Freezing immunoglobulins to see them move.

The issue of protein dynamics and its implications in the biological function of proteins are arousing greater and greater interest in different fields of molecular biology. In cryo-electron tomography experiments one may take several snapshots of a given biological macromolecule. In principle, a large enough collection of snapshots of the molecule may then be used to calculate its equilibrium configuration in terms of the experimentally accessible degrees of freedom and, hence, to estimate its potential energy. This information would be crucial in order to analyze the biological functions of biomolecules by directly accessing the relevant dynamical indicators. In this article, we analyze the results of cryo-electron tomography experiments performed on monoclonal murine IgG2a antibodies. We measure the equilibrium distribution of the molecule in terms of the relevant angular coordinates and build a mechanical model of the antibody dynamics. This approach enables us to derive an explicit expression of the IgG potential energy. Furthermore, we discuss the configuration space at equilibrium in relation to results from other techniques, and we set our discussion in the context of the current debate regarding conformation and flexibility of antibodies.

In situ transcription and splicing in the Balbiani ring 3 gene.

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co-transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20-25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi-intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well-defined unit in situ and that the C-terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation.

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.

Structural interaction between the nuclear pore complex and a specific translocating RNP particle.

The transport of Balbiani ring (BR) premessenger RNP particles in the larval salivary gland cells of the dipteran Chironomus tentans can be followed using electron microscopy. A BR RNP particle consists of an RNP ribbon bent into a ringlike structure. Upon translocation through the nuclear pore complex (NPC), the ribbon is straightened and enters the central channel of the NPC with the 5' end of the transcript in the lead. The translocating ribbon is likely to interact with the central channel but, in addition, the remaining portion of the ribbon ring makes contact with the periphery of the NPC. To determine the nature of this latter interaction, we have now studied the connections between the RNP particle and the border of the NPC during different stages of translocation using electron microscope tomography. It was observed that the 3' terminal domain of the ribbon always touches the nuclear ring of the NPC, but the precise area of contact is variable. Sometimes also a region on the opposite side of the ribbon ring reaches the nuclear ring. The pattern of contacts could be correlated to the stage of translocation, and it was concluded that the particle-nuclear ring interactions reflect a rotation of the ribbon ring in front of the central channel, the rotation being secondary to the successive translocation of the ribbon through the channel. The particle's mode of interaction with the NPC suggests that the initial contact between the 5' end domain of the ribbon and the entrance to the central channel is probably crucial to accomplish the ordered translocation of the premessenger RNP particle through the NPC.

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.

The crystal structure of the iron-free cytochrome c peroxidase and its implication for the enzymatic mechanism.

We report the refined structure of an iron-free form of cytochrome c peroxidase (CcP) at 2.3 A resolution. The backbone comparison between native CcP and iron-free CcP shows that the two structures have the same protein fold within experimental error. The only difference noted is in the heme pocket where the distance between the proximal histidine and the center of the protoporphyrin has increased. The results show that the iron-free CcP should be a good substitute for native CcP in fluorescence studies and thus also validate previous studies using iron-free CcPs as efficient fluorescent probes in electron transfer studies.

The central region of the synaptonemal complex in Blaps cribrosa studied by electron microscope tomography.

The synaptonemal complex (SC) in the beetle Blaps cribrosa contains a highly organized central element (CE), two flanking lateral elements (LEs), and a number of regularly spaced transverse filaments (TFs) crossing the central region. The CE is built like a ladder with two longitudinal components running in parallel and a number of regularly spaced transverse CE components, bridging the two longitudinal components. The CE is multi-layered with the ladders of the individual layers more or less in register. Essentially every TF originates in one of the LEs, crosses the CE through a transverse CE component and reaches the opposite LE; every transverse CE component in a given layer corresponds to one, and only one, TF. In a CE layer, short irregular pillars form the junctions between the transverse and longitudinal CE components. Adjacent pillars are connected to each other by fine fibrous bridges: the two pillars in the same transverse CE component are linked, and so are the pillars along each longitudinal component, and also more occasionally adjacent pillars in separate CE layers. It is proposed that a TF with the two associated short pillars represents the structural unit in the central region. The ordered structure of the CE is accomplished by linking adjacent pillars to each other into the well-defined three-dimensional organization of the CE.

The three-dimensional structure of the central region in a synaptonemal complex: a comparison between rat and two insect species, Drosophila melanogaster and Blaps cribrosa.

The highly ordered central region of the synaptonemal complex (SC) in Blaps cribrosa has recently been studied by electron microscope tomography (EMT), and a simple three-dimensional model presented. Using the same experimental approach we have now compared the central region in Blaps with the central regions in Drosophila melanogaster and rat. In all three species, the SCs exhibit a central element (CE) flanked by two lateral elements (LEs). The central region between the two LEs is crossed by transverse filaments (TFs). The Blaps CE element is the most ordered one with a well-defined ladder-like structure with two longitudinal components bridged by a number of regularly spaced transverse components, the rungs of the ladder. At the junctions between the longitudinal and transverse components there are prominent dense structures. The CE is multi-layered with the ladders of the separate layers in approximate register. In Drosophila the transverse CE components are as distinct and well organized as in Blaps, while in rat they are present but are less frequent and less well ordered. The longitudinal CE components in Drosophila are often fragmented and even more so in rat. The tomographic analysis revealed that in all three species the central region contains the same structural units: a single TF associated with two short pillars (or globules), which correspond to the junction structures. A fibrous lattice connects the two pillars/globules on the same TF forming the transverse CE component and those on adjacent TFs forming the longitudinal CE component; fibers between pillars/globules also link consecutive CE layers together. In the longitudinal component the number of fibrous bridges between the pillars/globules is related to the conspicuousness of the longitudinal component, i.e. Blaps has most, Drosophila almost as many, and rat considerably fewer bridges. We conclude that the central region in rat, Drosophila and Blaps contains the same basic structural unit but the degree of order and concentration of the units differ: a higher density seems to be accompanied by a higher order within the CE.

The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: a comparison with conventional techniques using electron microscope tomography.

A specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption-stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic-embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption-stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7-nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption-stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic-embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA-embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt-series to be taken for electron microscope tomography.

Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography.

A specific premessenger ribonucleoprotein (RNP) particle in the salivary glands of the dipteran Chironomus tentans was studied with electron microscope tomography during translocation from the cell nucleus to the cytoplasm. The RNP particle consists of a thin RNP fiber tightly folded into a ribbon, which is bent into a ring-like structure. Upon translocation through the pore, the particle is first orientated in a specific manner at the pore entrance, and subsequently the bent ribbon is gradually straightened and transported through the pore with the 5' end of the RNA in the lead. Concomitantly, the elementary RNP fiber constituting the ribbon is gradually unpacked and will appear more or less extended on the cytoplasmic side of the pore complex. The ordered nature of the process suggests a specific recognition of the RNP particle at the nuclear pore.

Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle.

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.

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.