The monomeric, tetrameric, and fibrillar organization of Fib: the dynamic building block of the bacterial linear motor of Spiroplasma melliferum BC3.

Spiroplasmas belong to the class Mollicutes, representing the minimal, free-living, and self-replicating forms of life. Spiroplasmas are helical wall-less bacteria and the only ones known to swim by means of a linear motor (rather than the near-universal rotary bacterial motor). The linear motor follows the shortest path along the cell's helical membranal tube. The motor is composed of a flat monolayered ribbon of seven parallel fibrils and is believed to function in controlling cell helicity and motility through dynamic, coordinated, differential length changes in the fibrils. The latter cause local perturbations of helical symmetry, which are essential for net directional displacement in environments with a low Reynolds number. The underlying fibrils' core building block is a circular tetramer of the 59-kDa protein Fib. The fibrils' differential length changes are believed to be driven by molecular switching of Fib, leading consequently to axial ratio and length changes in tetrameric rings. Using cryo electron microscopy, diffractometry, single-particle analysis of isolated ribbons, and sequence analyses of Fib, we determined the overall molecular organization of the Fib monomer, tetramer, fibril, and linear motor of Spiroplasma melliferum BC3 that underlies cell geometry and motility. Fib appears to be a bidomained molecule, of which the N-terminal half is apparently a globular phosphorylase. By a combination of reversible rotation and diagonal shift of Fib monomers, the tetramer adopts either a cross-like nonhanded conformation or a ring-like handed conformation. The sense of Fib rotation may determine the handedness of the linear motor and, eventually, of the cell. A further change in the axial ratio of the ring-like tetramers controls fibril lengths and the consequent helical geometry. Analysis of tetramer quadrants from adjacent fibrils clearly demonstrates local differential fibril lengths.

Globular tetramers of beta(2)-microglobulin assemble into elaborate amyloid fibrils.

Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-beta-strands and have revealed some details of local beta-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of three-dimensional structures of amyloid fibrils formed from full-length beta(2)-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold.

The flagellar filament structure of the extreme acidothermophile Sulfolobus shibatae B12 suggests that archaeabacterial flagella have a unique and common symmetry and design.

Archaea, constituting a third domain of life between Eubacteria and Eukarya, characteristically inhabit extreme environments. They swim by rotating flagellar filaments that are phenomenologically and functionally similar to those of eubacteria. However, biochemical, genetic and structural evidence has pointed to significant differences but even greater similarity to eubacterial type IV pili. Here we determined the three-dimensional symmetry and structure of the flagellar filament of the acidothermophilic archaeabacterium Sulfolobus shibatae B12 using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Processing of the cryo-negatively stained filaments included analysis of their helical symmetry and subsequent single particle reconstruction. Two filament subunit packing arrangements were identified: one has helical symmetry, similar to that of the extreme halophile Halobacterium salinarum, with ten subunits per 5.3 nm repeat and the other has helically arranged stacked disks with C(3) symmetry and 12 subunits per repeat. The two structures are related by a slight twist. The S. shibatae filament has a larger diameter compared to that of H. salinarum, at the opposite end of the archaeabacterial phylogenetic spectrum, but the basic three-start symmetry and the size and arrangement of the core domain are conserved and the filament lacks a central channel. This similarity suggests a unique and common underlying symmetry for archaeabacterial flagellar filaments.

Exploring the architecture of the intact supraspliceosome using electron microscopy.

Splicing of pre-mRNA takes place on a massive macromolecular machine in the nucleus of eukaryotic cells, the supraspliceosome. This particle is a multicomponent biological complex of RNA and proteins. It is composed of four sub-structures termed native spliceosomes that splice pre-mRNA. The structure of the native spliceosome, determined by cryo-EM at 20 A resolution, showed that it is composed of two distinct subunits. Previously, medium resolution structural analysis of supraspliceosomes by electron tomography was performed, yet little is known of how the native spliceosomes are arranged within the intact particle. To address this question the native spliceosomes were analyzed and reconstructed in the context of the intact particle, using electron microscopy combined with image processing. Good correlation was obtained between the structure of the isolated native spliceosome, solved by cryo-EM, and the native spliceosome within the intact supraspliceosome. An ordered assembly was revealed with different potential roles assigned to the small and large subunits of the native spliceosome. The edges of the small subunits, which are in the center of the supraspliceosome, form a right angle and thus facilitate close contacts between the small subunits generating a 4-fold pattern. The analysis of sub-complex orientation within the particle suggests a possible route within the supraspliceosome for the passage of pre-mRNA, which is known to hold the particle together.

The archaeabacterial flagellar filament: a bacterial propeller with a pilus-like structure.

Common prokaryotic motility modes are swimming by means of rotating internal or external flagellar filaments or gliding by means of retracting pili. The archaeabacterial flagellar filament differs significantly from the eubacterial flagellum: (1) Its diameter is 10-14 nm, compared to 18-24 nm for eubacterial flagellar filaments. (2) It has 3.3 subunits/turn of a 1.9 nm pitch left-handed helix compared to 5.5 subunits/turn of a 2.6 nm pitch right-handed helix for plain eubacterial flagellar filaments. (3) The archaeabacterial filament is glycosylated, which is uncommon in eubacterial flagella and is believed to be one of the key elements for stabilizing proteins under extreme conditions. (4) The amino acid composition of archaeabacterial flagellin, although highly conserved within the group, seems unrelated to the highly conserved eubacterial flagellins. On the other hand, the archaeabacterial flagellar filament shares some fundamental properties with type IV pili: (1) The hydrophobic N termini are largely homologous with the oligomerization domain of pilin. (2) The flagellin monomers follow a different mode of transport and assembly. They are synthesized as pre-flagellin and have a cleavable signal peptide, like pre-pilin and unlike eubacterial flagellin. (3) The archaeabacterial flagellin, like pilin, is glycosylated. (4) The filament lacks a central channel, consistent with polymerization occurring at the cell-proximal end. (5) The diameter of type IV pili, 6-9 nm, is closer to that of the archaeabacterial filament, 10-14 nm. A large body of data on the biochemistry and molecular biology of archaeabacterial flagella has accumulated in recent years. However, their structure and symmetry is only beginning to unfold. Here, we review the structure of the archaeabacterial flagellar filament in reference to the structures of type IV pili and eubacterial flagellar filaments, with which it shares structural and functional similarities, correspondingly.

Elongated oligomers assemble into mammalian PrP amyloid fibrils.

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly alpha-helical state into beta-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a beta form of recombinant mouse PrP (residues 91-231). The beta-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrP Sc. Using electron microscopy and image processing, we obtained three-dimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of approximately 60 A. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-beta amyloid fold.

The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: recovering high-resolution details from a flexible helical assembly.

Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the alpha-helices in domain D0 and the triple alpha-helical bundles in domain D1 at a resolution of 1/7A(-1). Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.

The structure of the helically perturbed flagellar filament of Pseudomonas rhodos: implications for the absence of the outer domain in other complex flagellins and for the flexibility of the radial spokes.

Bacterial flagella, the organelles of motility, are commonly divided into two classes: 'plain' and 'complex'. The complex filaments are pairwise, helically perturbed forms of the plain filaments and have been reported to occur only in Rhizobium and Pseudomonas. Previously, we reconstructed and analysed the structure of the complex filaments of Rhizobium lupini H13-3 and determined their unique symmetry and origin of the perturbations (Trachtenberg et al., 1986, J Mol Biol 190: 569-576; 1987, 195: 603-620; 1998, 276: 759-773; Cohen-Krausz and Trachtenberg, 1998, J Struct Biol 122: 267-282). Here, we analyse the structure of the flagellar filament of the other known complex filament, that of Pseudomonas rhodos, as reconstructed from electron microscope images. Compared with the filament of R. lupini, the filament of P. rhodos is more flexible, as implied from high-intensity darkfield light microscopy and, although constructed from flagellins of higher molecular weights (59 versus 41 kDa), has similar symmetry. Using cryonegative stained specimens and low-dose, field emission electron microscopy, we reconstructed and averaged 158 filaments each containing 170 statistically significant layer lines. The three-dimensional density maps of P. rhodos clearly suggest, when compared with those of R. lupini and the right-handed Salmonella typhimurium SJW1655, that R. lupini is missing the outer flagellin domain (D3), that the interior of the complex filament is rather similar to that of the plain filament and that the radial spokes (connecting domains D0 and D1), present in individual density maps, average out because of their variability and implied flexibility. Extending the three-start grooves and ridges on the propeller's surface, in the form of an Archimedean screw, may further improve the motility of the cell in viscous environments.

The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili.

Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g. reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition. Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion. The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin. The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments. The polymerization mode and small diameter may indicate the absence of a central channel in the filament. Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A. The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1. The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell. There is no evidence for a central channel. On the basis of the homology with the oligomerization domain of type IV pilin and the density distribution of the filament map, we propose a structure for the central core.