Comparison of single-particle analysis and electron tomography approaches: an overview.

Three-dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X-ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three-dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single-particle analysis. The first one is currently used to elucidate the three-dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.

Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study.

Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 A and 19 A, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.

Sepia officinalis hemocyanin: A refined 3D structure from field emission gun cryoelectron microscopy.

The extracellular respiratory pigment of the cuttlefish Sepia officinalis was observed by cryoelectron microscopy with conventional LaB(6) and field emission gun electron sources at 100 and 200 kV, respectively. Each image series was used to compute one 3D reconstruction volume with correction of the contrast transfer function by Wiener filtering. A strong boosting of the contrast was corrected by band-pass filtering of the final volumes, and a qualitative gain in resolution was observed when using the field emission gun electron microscope. In this volume, a strong signal is present down to 1/18 A(-1) and some meaningful information is obtained down to 1/12.5 A(-1). The complex is composed of five pairs of polypeptide chains and resembles a hollow cylinder with five wall oblique units and five inner arches. Three types of wall-wall connections termed pillar P1 to P3 are visible in this volume and the four functional units present in the arches are each linked to the wall by two arch-wall connections. The dispositions of the functional units in the arches of Sepia and Octopus hemocyanins share no common feature.

Three-dimensional architecture of human alpha 2-macroglobulin transformed with methylamine.

A frozen-hydrated sample embedded in vitreous ice of human alpha 2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and inter-subunit connections are also discussed.

Three-dimensional reconstruction from a frozen-hydrated specimen of the chiton Lepidochiton sp. hemocyanin.

A frozen-hydrated specimen of the hemocyanin of the chiton Lepidochiton sp. has been subjected to a three-dimensional reconstruction by the random conical tilt- series m wall and a collar complex. The wall is composed of five oblique wall units, disposed as a five-stranded, right-handed helix, separated by five clefts. The oblique wall unit is composed of two strings of functional units separated by a groove parallel with the cleft. The collar complex is a crown-like structure composed of five collar complex units, located at one end of the molecule and slightly protruding outside the cylinder wall. The collar complex unit comprises a collar unit probably composed of two functional units, one of which is connected to the wall, and an arch composed of two additional functional units, each connected to the wall by a narrow bridge. Each arch crosses a cleft between adjacent oblique wall units. The indentations present on both circular faces of the molecule and the dispositions of the masses resemble those of cephalopod hemocyanins.

Three-dimensional reconstruction of Lumbricus terrestris hemoglobin at 22 A resolution: intramolecular localization of the globin and linker chains.

A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terrestris was carried out by the 3D projection alignment method from electron microscopy images of a frozen-hydrated specimen at 22 A resolution. The results were analyzed by a new approach taking into account the evolution of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D6point-group symmetry is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex). The 12 globin subunits of each HGS are distributed around local 3-fold axis in four layers of three subunits. The first layer, the most external, contains monomeric globin chains 2A, 3A, and 5A. The three trimers corresponding to the nine remaining subunits have one subunit in each of the second (2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distances between the centers of the globin chains forming the trimers are in the ranges 20-32 A and 45-52 A. The linker complex is made up of two types of linking units. The first type forms three loops connecting globin chains of the second, third and fourth layers. The average molecular mass (Mm) of these subunits was 25 kDa. The second type forms the central structure, termed hexagonal toroid, and its 12 connections to the HGS. This structure corresponds to a hexamer of a single linking unit with a Mm (31.2 kDa), size and a shape different from those of the HGS loops. A careful study of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal layers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker chains belonging to two different types. We also propose a simple assembly sequence for the construction of HBL Hbs based on the architecture model.

Three-dimensional reconstruction of the alpha D and beta C-hemocyanins of Helix pomatia from frozen-hydrated specimens.

The three-dimensional (3D) reconstructions of the di-decameric forms of alpha D and beta C-hemocyanins of the Roman snail Helix pomatia and of the decameric half molecules of alpha D-hemocyanin were carried out on frozen-hydrated specimens observed in the electron microscope by using the random conical tilt series method. The three 3D volumes were examined by computing solid-body surface representations and slices through the volume and by eroding the structure progressively through raising of the threshold. The di-decameric molecule of alpha D and beta C-hemocyanins, reconstructed from side views, are very similar and are composed of a cylindrical wall, comprising ten oblique wall units, and of two collar complexes located at both ends of the cylinder, comprising each five arches and an annular collar made up of five collar units. Erosion of the structure reveals that the wall looks like a segment of a five-stranded right-handed helix and that each oblique wall unit resembles a figure 8 inclined to the right. The decameric half molecule of alpha D-hemocyanin, reconstructed from end-on views, resembles the whole molecule, except that the collar is thinner and appears composed of five independent collar complex units. It is suggested that the difference in structural appearance of the collar complex between the whole and the half alpha D-hemocyanin may be due to the missing cone artifact, induced by the angular limitations imposed by the goniometer of the electron microscope. The comparison between the alpha D-hemocyanin and the beta C-di-decameric hemocyanin at high thresholds suggests that in the beta C-hemocyanin the oblique wall units of each half molecule may be linked by two connections, whereas in alpha D-hemocyanin there may be only one. This difference in the number of connections may be responsible for the lower stability of the alpha D molecule at high salt concentration.

Three-dimensional reconstruction of Limulus polyphemus hemocyanin from cryoelectron microscopy.

Hemocyanin (Hc) the respiratory pigment of the horseshoe crab Limulus polyphemus (Lp) is composed of 48 approximately 75 kDa copper-containing subunits arranged in eight hexameric groups. In this study, we used the random conical tilt series method to do a three-dimensional (3D) reconstruction of Lp Hc observed in vitreous ice. This approach allowed the unambiguous determination of the handedness of the molecule. Lp Hc contains two superimposed 4 x 6mer structures possessing the same structural features as the other 4 x 6meric Hcs, namely flip and flop views and a rocking effect. Moreover, 3D fitting of the X-ray structure of subunit LpII with the reconstruction volume shows that the intra4 x 6meric contacts described in arthropod Hcs also occur within Limulus Hc. The two half-molecules composing the 8 x 6mer have their flop faces in contact (flop/flop association), the main links being formed by subunits LpIV. Model building shows that the flop/flop association is the only possible arrangement which allows the assembly of the whole particle. The two alternate constructions (flip/flop and flip/flip) are forbidden because of steric hindrance.

Three-dimensional reconstruction of native Androctonus australis hemocyanin.

A sample of native 4 x 6-meric hemocyanin of Androctonus australis was negatively stained with the double-layer technique, and was observed by transmission electron microscopy under low-dose conditions with a 50 degree and 0 degree tilt. The three-dimensional reconstruction method from "Single-exposure, random conical tilt series" was then applied. Independent three-dimensional reconstructions were obtained from the top, side and 45 degree views. Despite a pronounced flattening effect, presumably due to the specimen preparation technique, the positions of the 24 subunits composing the oligomer were unequivocally determined. This experiment definitely solves the problem of the architectural organization of the subunits in the cheliceratan 4 x 6-meric hemocyanins. Moreover, distinction between the flip and flop faces and an attenuated rocking effect were observed.

Intramolecular localization of the functional units of Sepia officinalis hemocyanin by immunoelectron microscopy.

The quaternary structure of Sepia officinalis hemocyanin (Hc) as studied in immunoelectron microscopy with rabbit IgGs and Fab fragments raised against functional units (FU) Soc, Sod, Soe, Sof, Sog, and Soh and fragment Soab. The architecture of immunocomplexes shows that (i) epitopes characteristic of FUs Soc and Sog and of fragment Soab are located in the two external tiers of FUs, (ii) FUs Soh and Soe or Sod are located in arches. These results were confirmed using immunocomplexes made up of Sepia Hc and IgGs or Fab fragments purified from antisera raised against FUs of Octopus vulgaris and Octopus dofleini. Frozen-hydrated immunocomplexes containing one Hc molecule and at least one FU-specific Fab fragment were observed in the electron microscope and submitted to image processing. When the Hc molecule is viewed along its 5-fold axis (i) anti-Soc Fab fragments project on a radius passing through the arch's pillar, (ii) anti-Sof Fabs project slightly out of the arches, and (iii) anti-Soh Fabs project between neighboring arches. When applied to a recent three-dimensional (3D) reconstruction volume, these results allow us to deduce the intramolecular location of five of the eight FUs. For the last three FUs limited uncertainties remain: (i) Soc can be located in two positions in the external tier of FUs; (ii) Soa and Sob can both occupy three positions in the external tiers; and (iii) because of an immunological cross-reactivity Sod may be located in the wall and Soe in the arch, or vice versa. An analysis of the quaternary structure considering the possible locations of the 80 FUs and postulating a single type of subunit shows that 80 possibilities of paths still exist for the polypeptide chain. To solve definitely these 80 possibilities only five questions remain to be answered.

Quaternary structure of Octopus vulgaris hemocyanin. Three-dimensional reconstruction from frozen-hydrated specimens and intramolecular location of functional units Ove and Ovb.

A frozen-hydrated sample of Octopus vulgaris hemocyanin was imaged at 0 degree and 40 degrees tilt angle under low dose conditions by transmission electron microscopy. A three-dimensional reconstruction by the method of random conical tilt series produced a three-dimensional volume to which a D5 symmetry was applied. Examination of serial sections in the volume and surface representation at various thresholds allowed the five arches containing functional unit Ovg to be localized at the interdimeric subunit groove. In another set of experiments specific polyclonal antibodies were used to label functional units Ovb and Ove in the cylinder wall. The observation of the negatively stained immunocomplexes showed that Ovb is located in the external tiers of functional units and Ove in the internal tier. These results suggest that the direction of the polypeptide chains in the cylinder wall may be only partially antiparallel. A model of the quaternary structure is proposed with the following features: (1) the external tiers of functional units comprise four units each (Ova-d) coming from a single polypeptide chain; (2) the internal tier comprises two functional units from each polypeptide chain (Ove-f); (3) the interdimeric subunit arches connect the two copies of a single functional unit (Ovg) located in each polypeptide chain.

Three-dimensional reconstruction of the chlorocruorin of the polychaete annelid Eudistylia vancouverii.

The chlorocruorin of the polychaete Eudistylia vancouverii observed in the electron microscope in vitreous ice, was subjected to a three-dimensional (3D) reconstruction by the random conical tilt series method. The 3D volume with a resolution of 35 A reconstructed from 1062 images in top, side and intermediate view orientations has a D6 point-group symmetry. It possesses the characteristic hexagonal bilayer (HBL) appearance. Each hexagonal half-molecule comprises size hollow globular substructures (HGS) presumed to correspond to the dodecameric subunits. In projection, when the molecule is viewed along its 6-fold axis, the two halves are not perfectly eclipsed. The vertices of the upper hexagonal layer are 14 degrees rotated clockwise compared with those of the lower half. At a threshold displaying 100% of the expected molecular volume, the 3D volume contains in its center a flat hexagonal central mass disconnected from the rest of the volume. Several types of connections, termed c1 through c4, are visible between the HGSs. The c1 and c2 connections link the HGSs of the same hexagonal half-molecule. The c3 connections make a hexagonal inner bracelet linking the HGSs of each half-molecule. The c4 connections link pairs of HGSs superposed in the two hexagonal layers. Because of the half-molecules rotation around the 6-fold axis, the two HGSs linked by a c4 connection are not exactly superposed. It is proposed that the c3 and c4-connection bodies and less probably the flat central hexagonal mass are composed of chloroheme-deficient linker chains. When eroding the 3D volume by raising the threshold, the HGS appears composed of three elongated structures likely containing four globin chains. In addition, they show an approximate 3-fold symmetry. At high thresholds, two of these masses, dumbbell-shaped, separate into globular masses while the third structure remains compact as long as 1% of the molecular volume is displayed.

Strong expression of GFAP mRNA in rat hippocampus after a closed-head injury.

We investigated the spatiotemporal GFAP mRNA expression over a period of 11 days following brain injury in rats caused by impact acceleration, which is known to produce diffuse axonal injury (DAI). We observed widespread GFAP mRNA expression throughout the brain, which was more rapid and intense in the hippocampus. This expression was obvious in most animals 2 days after injury and appeared maximal at day 6. Although it decreased by day 11, the level of expression remained high compared with control levels. We noted slight differences in time of onset and the magnitude of the response between hippocampus and white matter structures or cortical areas. The different mechanisms able to trigger this response are discussed in regard to histopathological changes observed in DAI models.

Innervation of the caudal denervated ventral roots and their target muscles by the rostral spinal motoneurons after implanting a nerve autograft in spinal cord-injured adult marmosets.

OBJECT: The authors conducted a study to determine the effects of using a nerve autograft (NAG) to promote and guide axonal regrowth from the rostral spinal cord to the caudal lumbar ventral nerve roots to restore hindlimb motor function in adult marmosets after lower thoracic cord injury. METHODS: Nine animals underwent a left-sided hemisection of the spinal cord at T-12 via left-sided T9-L3 hemilaminectomy, with section of all ipsilateral lumbrosacral ventral nerve roots. In the experimental group (five animals), an NAG obtained from the right peroneal nerve was anastomosed with the sectioned and electrophysiologically selected lumbar ventral roots (left L-3 and L-4) controlling the left quadriceps muscle and then implanted into the left ventrolateral T-10 cord. In the control group (four animals), the sectioned/selected lumbar ventral roots were only ligated. After surgery, all marmosets immediately suffered from complete paralysis of their left hindlimb. Five months later, some clinical signs of reinnervation such as tension and resistance began to appear in the paralyzed quadriceps of all experimental animals that received autografts. Nine months postoperatively, three of the five experimental marmosets could maintain their lesioned hindlimb in hip flexion. Muscle action potentials and motor evoked potentials were recorded from the target quadriceps in all experimental marmosets, but these potentials were absent in the control animals. Horseradish peroxidase retrograde labeling from the distal sectioned/reconnected lumbar ventral roots traced 234+/-178 labeled neurons in the ipsilateral T8-10 ventral horn, mainly close to the NAG tip. Histological analysis showed numerous regenerating axons in this denervated/reconnected nerve root pathway, as well as newly formed motor endplates in the denervated/reinnervated quadriceps. No axonal regeneration was detected in the control animals. CONCLUSIONS: These data indicate that the rostral spinal neurons can regrow into the caudal ventral roots through an NAG, thereby innervating the target muscle in adult marmosets after spinal cord injury.

[Horseradish peroxidase retrograde labeling of primary sensory axons in rats: a comparison between three different intraspinal injections methods]. / Marquage rétrograde des axones sensitifs primaires par la peroxydase du raifort chez le rat: mise au point de la technique d'injection intramédullaire.

STUDY AIM: In order to improve the results of intraspinal retrograde labeling of post-ganglionic primary sensory axons by horseradish peroxidase (HRP), the authors compared three different intraspinal injection methods of this tracer into the inferior thoracic spinal cord in the rat. MATERIAL AND METHOD: 'Open field' method (group 1, N = 8); stereotactic injection, needle tip diameter = 0.72 mm (group 2, N = 8); stereotactic injection, needle tip diameter = 0.24 mm (group 3, N = 8). Histological features of the spinal injection site showed that tissue damages due to injection was more extensive and deeper than expected. HRP transported in retrograde fashion from injection site to sensory body cells located in dorsal root ganglia (DRG) was revealed by the Mesulam histochemical technique. RESULTS: The mean number of labeled neurons per DRG was 652 in group 3, 116 in group 2, and 77 in group 1. Differences were statistically significant, especially between groups 1 and 3 (P = 4.10(-16)) and groups 2 and 3 (P = 2.10(-17)). CONCLUSION: Retrograde labeling of primary sensory axons by HRP (or another axonal tracer) with fine needle stereotactic intraspinal injection may represent an alternative to anterograde labeling. This reliable and reproducible method may be useful in studies dealing with regeneration of post-ganglionic primary sensory axons.

Preservation of protein globules and peptidoglycan in the mineralized cell wall of nitrate-reducing, iron(II)-oxidizing bacteria: a cryo-electron microscopy study.

Iron-oxidizing bacteria are important actors of the geochemical cycle of iron in modern environments and may have played a key role all over Earth's history. However, in order to better assess that role on the modern and the past Earth, there is a need for better understanding the mechanisms of bacterial iron oxidation and for defining potential biosignatures to be looked for in the geologic record. In this study, we investigated experimentally and at the nanometre scale the mineralization of iron-oxidizing bacteria with a combination of synchrotron-based scanning transmission X-ray microscopy (STXM), scanning transmission electron microscopy (STEM) and cryo-transmission electron microscopy (cryo-TEM). We show that the use of cryo-TEM instead of conventional microscopy provides detailed information of the successive iron biomineralization stages in anaerobic nitrate-reducing iron-oxidizing bacteria. These results suggest the existence of preferential Fe-binding and Fe-oxidizing sites on the outer face of the plasma membrane leading to the nucleation and growth of Fe minerals within the periplasm of these cells that eventually become completely encrusted. In contrast, the septa of dividing cells remain nonmineralized. In addition, the use of cryo-TEM offers a detailed view of the exceptional preservation of protein globules and the peptidoglycan within the Fe-mineralized cell walls of these bacteria. These organic molecules and ultrastructural details might be protected from further degradation by entrapment in the mineral matrix down to the nanometre scale. This is discussed in the light of previous studies on the properties of Fe-organic interactions and more generally on the fossilization of mineral-organic assemblies.

Fast, robust, and accurate determination of transmission electron microscopy contrast transfer function.

Transmission electron microscopy, as most imaging devices, introduces optical aberrations that in the case of thin specimens are usually modeled in Fourier space by the so-called contrast transfer function (CTF). Accurate determination of the CTF is crucial for its posterior correction. Furthermore, the CTF estimation must be fast and robust if high-throughput three-dimensional electron microscopy (3DEM) studies are to be carried out. In this paper we present a robust algorithm that fits a theoretical CTF model to the power spectrum density (PSD) measured on a specific micrograph or micrograph area. Our algorithm is capable of estimating the envelope of the CTF which is absolutely needed for the correction of the CTF amplitude changes.