JoséL. Carrascosa

General morphology and capsid fine structure of African swine fever virus particles.

The structure of African swine fever virus particles has been examined by electron microscopy. The analysis of virions prepared by negative staining, thin sectioning, and freeze-drying and shadowing showed that the virus particle was composed of several concentric structures with an overall icosahedral shape. The inner region of the virus particles was a nucleoid that was surrounded by a membrane covered by the capsid. The capsid had side-to-side dimensions of 172 to 191 nm and was built up by capsomers arranged in an hexagonal lattice. Computer-filtered electron micrographs of either negatively stained or freeze-dried and shadowed capsids revealed capsomers with a hexagonal outline and a hole in the center. The intercapsomer distance ranged from 7.4 to 8.1 nm. The triangulation number of the capsid was estimated to be 189 to 217, indicative of 1892 to 2172 capsomers. Extracellular African swine fever virus particles had an external membrane that resembled the cytoplasmic unit membrane.

Three-dimensional reconstruction of the connector of bacteriophage φ29 at 1.8 nm resolution

The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1. These connectors are built up as dodecamers of only one structural polypeptide (p10). Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping. The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid. The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation). These features are compatible with an active role for the connector in the process of DNA packaging.

Structure of the head-tail connector of bacteriophage φ29

Abstract The head-to-tail connecting region of bacteriophage φ29 has been studied by isolating neck-tail complexes from disrupted phage. These complexes can be isolated with appendages (from wild-type phage) or without appendages (from phage mutant sus12 ). Treatment of the neck-tail complex without appendages with urea or guanidinium hydrochloride releases the tail protein (p9) from the neck complex (proteins p10 and p11). Electron micrographs of φ29 necks show that they are composed of two collars and a thin axial tube. There is an internal hole along the longitudinal axis, from the upper collar to the thin tube. Image-processing analysis of electron micrographs of two-dimensional crystals of necks shows that the neck of phage φ29 consists of 12 external units and an internal area of apparent 6-fold symmetry, with a hole in the centre.

Reversible interaction of beta-actin along the channel of the TCP-1 cytoplasmic chaperonin

The cytoplasm of eukaryotes contains a heteromeric toroidal chaperonin assembled from the t-complex TCP-1 and several other related polypeptides. The structure of the TCP-1 cytoplasmic chaperonin and that of the binary complex formed between this chaperonin and unfolded beta-actin have been studied using electron microscopy and image processing techniques. Two-dimensional averaging of front views reveals a circular stain-excluding mass surrounding a central stain-penetrating region in which the stain is excluded upon actin binding. Sections of a three-dimensional reconstruction of the chaperonin show that the inner core is an empty channel that becomes filled upon binary complex formation with unfolded beta-actin. Upon incubation with Mg-ATP, the beta-actin:chaperonin complex discharges the actin such that the chaperonin central cavity reappears. Side views from different forms of TCP-1 reveals that upon Mg-ATP binding, the cytoplasmic chaperonin undergoes a structural rearrangement that is confirmed using a new classification method.

Bacteriophage T3 connector: Three-dimensional structure and comparison with other viral head-tail connecting regions☆

The bacteriophage T3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates. A three-dimensional reconstruction of T3 connectors was obtained by collection of tilted views and using the direct Fourier method, up to 2.3 nm resolution. The reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions. The T3 connector has a height of about 10.9 nm, with two clearly defined domains: a wider one 14.4 nm in diameter, with 12 morphological units in the periphery, and a narrower one, 9.7 nm in diameter. There is a channel clearly defined in the narrower domain that almost closes along the wider domain. Comparison of the three-dimensional structure obtained for the connector of phages T3 and phi 29, and that of the neck extracted from phage phi 29 particles, reveals striking similarities and significant differences. A model for a general connector to account for the common functions carried out by these viral assemblies is discussed together with the possible role of the channel for DNA translocation.

Three-dimensional reconstruction of bacteriophage φ29 neck particles at 2.2 nm resolution☆

Abstract The three-dimensional structure of the head-to-tail connecting region of bacteriophage φ29 has been studied by analysing two-dimensional, hexagonal ordered aggregates of negatively stained viral necks to a resolution of 2.2 nm. These necks are composed of two proteins, p10 and p11; p10 being the connector protein. A 12-folded and a 6-folded axially symmetric domain are present in the specimen. The 12-folded domain is the larger part of the structure; it consists of 12 subunits associated in pairs. These subunits appear to be more closely paired towards the centre, where only six subunits are resolved forming the 6-folded domain. The pairs of subunits present an important twist between the 12-folded and the 6-folded areas. A conical hole is formed at the centre of the structure. This hole is more open at the 12-folded domain than at the level of the possible zone of interaction between p10 and p11, where it is almost closed. Protein p11 is very poorly represented in the reconstruction, probably due to lack of staining. The structure described for the φ29 neck has many of the attributes expected for an active device involved in bacteriophage DNA encapsidation.

Structural comparison of prokaryotic and eukaryotic chaperonins.

Chaperonins are key components of the cell machinery and are involved in the productive folding of proteins. Most chaperonins share a common general morphology based in a cylinder composed of two rings of 7-9 subunits, with a conspicuous cavity inside the particle. Chaperonins have been classified into two groups according to their sequence homologies: type I, whose better known member is GroEL, and type II comprising the eukaryotic cytosolic CCT and the archaebacterial thermosome, among others. Although the basic structure of both chaperonin types is rather similar, there are a number of basic differences among them. Whereas GroEL is rather non-specific regarding its substrate, CCT is more specialized, and plays a fundamental role in the folding of cytoskeletal proteins. Another important difference is that GroEL is an homopolymer, while CCT is an heteromeric complex built up of eight different polypeptides. Furthermore, GroEL requires a cofactor (GroES) that is not present in the type II chaperonins. Recent studies of the structure of CCT have allowed a deeper insight into its function. Electron microscopic analyses have revealed a different behavior of this chaperonin after binding to nucleotides, respect to GroEL. The atomic structure of the thermosome fits into the electron microscopy reconstructed volume of the CCT. This fitting gives clues to compare the structural transitions of GroEL and CCT during the folding cycle. The different changes undergone by the two chaperonins suggest the existence of differences in the way they bind substrates and enlarge the internal cavity, as well as a different type of signaling between the two rings of the types I and II chaperonins.

Three-dimensional structure of T3 connector purified from overexpressing bacteria☆

The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein. The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens. A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens. In addition, single particle averaging techniques were used with other preparations. The average images obtained from these two approaches gave similar results. A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures. The resolution of the three-dimensional map was 1.65 nm. The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter. The height of the reconstructed connector has been determined to be around 8.5 nm. The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.

Glycosylated components of african swine fever virus particles

Extracellular African swine fever (ASF) virus particles were specifically agglutinated by several lectins, suggesting the presence of surface glycosylated component(s) containing at least glucose, mannose, or both; galactose, N-acetylgalactosamine, or both; N-acetylneuraminic acid and N-acetylglucosamine, but not fucose. When virions were purified from infected Vero cells labeled with [14C]glucosamine, [14C]galactose and analyzed by polyacrylamide gel electrophoresis, no major structural glycoproteins were detected. However, several species of glycolipids were found when virions were extracted with organic solvents and analyzed by thin layer chromatography. These, plus two minor glycosylated structural components, of apparent mol wt 230K and 95K, could account for the agglutination of ASF virions with concanavalin A.

Single Centrosome Manipulation Reveals Its Electric Charge and Associated Dynamic Structure

The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 +/- 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 10(3) electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca(2+), over the thermally induced dynamic structure of the centrosome.