Three-dimensional structure of the frozen-hydrated flagellar filament. The left-handed filament of Salmonella typhimurium

Electron micrographs of frozen-hydrated preparations of flagellar filaments of Salmonella typhimurium were used to obtain a three-dimensional reconstruction of the structure. The filaments were obtained from the mutant SJW1660, which produces straight, left-handed filaments. The subunits in this filament are thought to be all in the L-state. The structure consists of a set of 11 longitudinal segmented rods of density that lie at a radius of 70 A. The outermost feature of the filament is a set of knobs of density that project outward from the rods. The interior of the filaments consists of arms that extend inward radially from the segmented rods. The 11 segmented rods and their interconnections are noteworthy because current theories regarding filament structure involve switching of subunits between the L and R states co-operatively along the directions of the rods.     

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.     

Three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus. A flagellin lacking the outer domain and its amino acid sequence lacking an internal segment

We obtained a three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus CB15 from electron micrographs of negatively stained preparations. The C. crescentus filament appears, both in negative stain and in the frozen-hydrated state, significantly smoother and narrower than other filaments. Its helical symmetry, and unit cell size, however, are similar to that of other filaments. Although the molecular weight of the C. crescentus flagellin is about half that of other plain flagellins, there is only one monomer per unit cell as indicated by diffraction studies and by linear mass density measurements with the scanning transmission electron microscope. Alignment of the primary amino acid sequences of Salmonella typhimurium (serotype i) and C. crescentus (29,000 Mr) flagellins shows that whereas there is homology at the amino and carboxyterminal ends of the two sequences, the central segment of the S. typhimurium sequence has no homology to that of C. crescentus. A correlated comparison between the three-dimensional reconstructions of the two filaments and primary amino acid sequences of the two flagellins suggests that: (1) the C. crescentus subunit is missing the outer molecular domain but is, otherwise, similar to that of S. typhimurium; (2) the outer molecular domain in S. typhimurium corresponds, therefore, to a central stretch of the primary amino acid sequence; and (3) the outer molecular domain, missing in C. crescentus, is not obligatory for flagellar motility.     

General model of myosin filament structure. 3. Molecular packing arrangements in myosin filaments

Following the original proposals about myosin filament structure put forward as part of a general myosin filament model (Squire, 1971, 1972) it is here shown what the most likely molecular packing arrangements within the backbones of certain myosin filaments would be assuming that the model is correct. That this is so is already indicated by recently published experimental results which have confirmed several predictions of the model (Bullard and Reedy, 1972; Reedy et al., 1972; Tregear and Squire, 1973).The starting point in the analysis of the myosin packing arrangements is the model for the myosin ribbons in vertebrate smooth muscle proposed by Small &; Squire (1972). It is shown that there is only one reasonable type of packing arrangement for the rod portions of the myosin molecules which will account for the known structure of the ribbons and which is consistent with the known properties of myosin molecules. The dominant interactions in this packing scheme are between parallel myosin molecules which are related by axial shifts of 430 Å and 720 Å. In this analysis the myosin rods are treated as uniform rods of electron density and only the general features of two-strand coiled-coil molecules are considered.Since the general myosin filament model is based on the assumption that the structures of different types of myosin filament must be closely related, the packing scheme derived for the myosin ribbons is used to deduce the structures of the main parts (excluding the bare zones) of the myosin filaments in a variety of muscles. It is shown in each case that there is only one packing scheme consistent with all the available data on these filaments and that in each filament type exactly the same interactions between myosin rods are involved. In other words the myosin-myosin interactions involved in filament formation are specific, they involve molecular shifts of either 430 Å or 720 Å, and are virtually identical in all the different myosin filaments which have been considered. Apart from the myosin ribbons, these are the filaments in vertebrate skeletal muscle, insect flight muscle and certain molluscan muscles.In the case of the thick filaments in vertebrate skeletal muscle the form of the myosin packing arrangement in the bare zone is considered and a packing scheme proposed which involves antiparallel overlaps between myosin rods of 1300 Å and 430 Å. It is shown that this scheme readily explains the triangular profiles of the myosin filaments in the bare zone (Pepe, 1967, 1971) and many other observations on the form of these myosin filaments.Finally it is shown that the cores of several different myosin filaments, assuming they contain protein, may consist of different arrangements of one or other of two types of core subfilament.     

The rigidity of bacterial flagellar filaments and its relation to filament polymorphism.

We determined and correlated the rigidity of Salmonella typhimurium, Escherichia coli, and Rhizobium lupini flagellar filaments representing various structural and polymorphic states (plain, complex, straight, superhelical, and right- and left-handed). Persistence length, from which the filament's rigidity and other parameters (Young's modulus, bending force constant, buckling persistence length, flexural deformation, and flexural time) were derived, was determined from electron micrographs of isolated, negatively stained filaments. Outer diameters and radii of strong intersubunit connectivity were determined from three-dimensional image reconstructions and radial mass density profiles from scanning transmission electron microscopy. All filaments appear to be highly rigid with no evident correlation with their helical sense or superhelicity. The complex filament of R. lupini is rigid to the extent that it becomes brittle. The overall flexibility of the flagellum seems to stem mainly from the hook and not from the filament. Polymorphism is probably related to the propelling properties and hydrodynamic shape of the filament rather than to its rigidity.     

Automated segmentation of electron tomograms for a quantitative description of actin filament networks

Cryo-electron tomography allows to visualize individual actin filaments and to describe the three-dimensional organization of actin networks in the context of unperturbed cellular environments. For a quantitative characterization of actin filament networks, the tomograms must be segmented in a reproducible manner. Here, we describe an automated procedure for the segmentation of actin filaments, which combines template matching with a new tracing algorithm. The result is a set of lines, each one representing the central line of a filament. As demonstrated with cryo-tomograms of cellular actin networks, these line sets can be used to characterize filament networks in terms of filament length, orientation, density, stiffness (persistence length), or the occurrence of branching points.     

Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.     

Flagellar hook structures of Caulobacter and Salmonella and their relationship to filament structure

Native flagellar hooks from a polarly flagellated bacterium, Caulobacter crescentus, and polyhooks from a peritrichously flagellated bacterium, Salmonella typhimurium. have been studied by densitometry of electron micrographs of negatively stained specimens, followed by computerized Fourier analysis and three-dimensional reconstruction. The two structures are remarkably similar. In both cases, the subunits are arranged along a right-handed basic helix of 2.3 nm pitch with successive subunits separated by an azimuthal angle of 64 to 65 °, and there is a pronounced system of continuous 6-start grooves and ridges on the surface of the structures. The subunit of Salmonella (M_r 42,000, versus 70,000 for Caulobacter) is somewhat thinner and yields a smaller overall hook diameter. The “bent finger” subunit shape and orientation in both cases suggests that the hook could bend readily by a sliding motion in the 11-start direction at inner radii, with the 6-start groove preventing collision at outer radii. The basic helical pitch of the Salmonella hook structure, and the number of subunits per basic helical turn (5.56) makes it highly compatible with the Salmonella flagellar filament (2.6 nm pitch. 5.51 subunits per turn); so also does the elongated shape and tilt angle of the hook and flagellin subunits in the respective structures. The two structures may therefore conjoin directly in the intact flagellum, although participation of a minor protein is not ruled out by the data.     

The fine structure of the fertilization membrane of the feather star Comanthus japonica (Echinodermata: Crinoidea)

Scanning electron microscopy reveals that the outer surface of the Comanthus fertilization membrane bears a network of 14 µ high ridges outlining rows of polygonal facets; however, no spines are present. The so-called spines reported previously by light microscopists were simply optical cross sections of the ridges. Transmission electron microscopy reveals that the fertilization membrane has (1) an outer component consisting mainly of dense, granular material, and (2) an inner component consisting mainly of an interlacing network of 50 Å fibers of moderate electron density. Associated with the fibers of the inner component are dense granular rods and dense lentoid discs.     

Molecular architecture of the neurofilament. I. Subunit arrangement of neurofilament L protein in the intermediate-sized filament

Using the smallest subunit (NF-L) of a neurofilament and a glial fibrillary acidic protein, the subunit arrangement in intermediate filaments was studied by low-angle rotary shadowing. NF-L formed a pair of 70 to 80 nm rods in a low ionic strength solution at pH 6.8. Two 70 to 80 nm rods appeared to associate in an antiparallel manner with an overlap of about 55 nm, almost the same length as the alpha-helix-rich central rod domain of intermediate filament proteins. The overlap extended for three-beaded segments, present at 22 nm intervals along the pairs of rods. The observations that (1) 70 to 80 nm rods were a predominant structure in a low ionic strength solution at pH 8.5, (2) the molecular weights of the rod and the pair were measured by sedimentation equilibrium as 190,000 and 37,000 respectively, and (3) the rods formed from the trypsin-digested NF-L had a length of about 47 nm, indicated that the 70 to 80 nm rod is the four-chain complex and the pair of rods is the eight-chain complex. Similar structures were observed with glial fibrillary acidic protein, indicating that these oligomeric structures are common to other intermediate filament proteins. NF-L assembled into short intermediate-sized filaments upon dialysis against a low-salt solution containing 1 to 2 mM-MgCl2 at 4 degrees C. The majority of these short filaments possessed four or five-beaded segments, suggesting that the pair of rods were arranged in a half-staggered fashion in neurofilaments. On the basis of these observations, we propose the following model for the intermediate filament subunit arrangement. (1) The four-chain complex is the 70 to 80 nm rod, in which two coiled-coil molecules align in parallel and in register. (2) Two four-chain complexes form the eight-chain complex by associating in an antiparallel fashion with the overlap of the entire central rod domain. (3) The eight-chain complex is the building block of the intermediate filament. The eight-chain complexes are arranged in a half-staggered fashion within the intermediate filament.     

Rigor crossbridge structure in tilted single filament layers and flared-X formations from insect flight muscle

The averaged structure of rigor crossbridges in insect flight muscle has been studied in filtered images. Their three-dimensional structure has been deduced by relating tilt views of single filament layers in 25 nm longitudinal sections (myac layers and actin layers) to the flared-X appearance in 15 nm cross-sections showing single crossbridge levels. Tilting myac or actin layers around the filament axis makes crossbridges show one of two patterns. Beadlike densities appear either singly over thin filaments ("center-beading") or doubled and flanking thin filaments ("straddle-beading"). These express two different projections from the crossbridge-actin complexes as seen end-on in flared-X formations. Tannic acid/glutaraldehyde fixation gave improved actin preservation, showing, in 15 nm cross-sections, the long-pitch helical strands as "two-dot" profiles of consistent azimuth in the gaps between double chevrons. The azimuth in the flared-X arms was then inferred from lattice relationships, since it was not seen directly. The tangential attachment of comma-shaped crossbridges to the inferred actin dyad fits the binding geometry in recent actin-subfragment 1 complex reconstructions. However, averaged crossbridge structure differs between lead and rear members of double chevrons, unlike the uniform heads on decorated actin. In filtered images of myac layers, the lead bridges are dense and steeply angled; the rear chevron is seen as a dense bead over the thin filament with faint, less angled bars extending laterally. Actin layer images also suggest that rear and lead bridges differ in angle. Left and right flared-X arms are end-on views of lead and rear chevron bridges, respectively, and differ in shape. Improved fixation with tannic acid/glutaraldehyde allows us to distinguish three crossbridge domains in flared-X arms: (1) a dense bulb-like head merged into the thin filament; (2) a dense but thinner neck tangential to actin; and (3) a faint thin stem joining the necks to myosin filaments. Shape differences in lead and rear members between the head-neck-actin complexes are indicated by the names "L sigmoid" and "R dogleg". Within crossbridges, internal angles between the head-neck axis and the head-actin-head axis differ between sigmoid and dogleg by about 30 degrees, implying a flexible junction between bridge-head and bridge-neck. Lead and rear bridges are axially at least 13 nm apart on actin; the expected 60 degrees difference in azimuth is expressed by head-neck portions, but the head-actin-head axis rotates by only 30 degrees.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructure of Mitochondria and Crystal-containing Bodies in Mature Ballistospores of the Fungus Basidiobolus ranarum as Revealed by Freeze-etching

By using the freeze-etch technique, a regular pattern on both sides of the outer mitochondrial membrane can be demonstrated in mature spores of Basidiobolus ranarum. Bands consisting of 5 to 10 parallel ridges, each of which is 20 nm wide, envelop the outer as well as the inner side of this membrane. Hexagonal crystals, probably representing stored protein, are enclosed in special bodies with a very smooth surrounding unit membrane. The crystals are formed by parallel rods which consist of globular subunits, 6.5 to 7.0 nm wide.     

The three-dimensional structure of the nemaline rod Z-band

In nemaline myopathy and some cardiac muscles, the Z-band becomes greatly enlarged and contains multiple layers of a zigzag structure similar to that seen in normal muscle. Because of the additional periodicity in the direction of the filament axis, these structures are particularly favorable for three-dimensional analysis since it becomes possible to average the data in all three dimensions and thus improve the reliability of the reconstruction. Individual views of the structure corresponding to tilted longitudinal and transverse sections were combined by matching the phases of common reflections. Examination of the tilted views strongly suggested that to the available resolution, the structure possesses fourfold screw symmetry along the actin filament axes. This symmetry could be used both in establishing the correct alignment for the combination of individual tilted views and to generate additional views not readily accessible in a single tilt series. The reconstruction shows actin filaments from one sarcomere surrounded by an array of four actin filaments with opposite polarity from the adjacent sacormere. The actin filaments show a right- handed twist and are connected by a structure that links adjacent filaments with the same polarity at the same axial level, then runs parallel to the filaments, and finally forms a link between two actin filaments whose polarity is opposite to that of the first pair. The connecting structure is probably composed of alpha-actinin which is located in Z-bands and cross-links actin filaments. The connecting structure may consist of two alpha-actinin molecules linking actin filaments of opposite polarity.     

The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model

Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F-actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly contributes to the outer part of the massive base. Quantitative evaluation of successive crossover spacings along individual F-actin filaments revealed the deviations from the mean repeat to be compensatory, i.e., short crossovers frequently followed long ones and vice versa. The variable crossover spacings and diameter of the F-actin filament together with the local unraveling of the two long-pitch helical strands are explained in terms of varying amounts of compensatory "lateral slipping" of the two strands past each other roughly perpendicular to the filament axis. This intrinsic disorder of the actin filament may enable the actin moiety to play a more active role in actin-myosin-based force generation than merely act as a rigid passive cable as has hitherto been assumed.     

Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo

The precise assembly of the highly organized filament systems found in muscle is critically important for its function. It has been hypothesized that nebulin, a giant filamentous protein extending along the entire length of the thin filament, provides a blueprint for muscle thin filament assembly. To test this hypothesis, we generated a KO mouse model to investigate nebulin functions in vivo. Nebulin KO mice assemble thin filaments of reduced lengths and approximately 15% of their Z-disks are abnormally wide. Our data demonstrate that nebulin functions in vivo as a molecular ruler by specifying pointed- and barbed-end thin filament capping. Consistent with the shorter thin filament length of nebulin deficient mice, maximal active tension was significantly reduced in KO animals. Phenotypically, the murine model recapitulates human nemaline myopathy (NM), that is, the formation of nemaline rods combined with severe skeletal muscle weakness. The myopathic changes in the nebulin KO model include depressed contractility, loss of myopalladin from the Z-disk, and dysregulation of genes involved in calcium homeostasis and glycogen metabolism; features potentially relevant for understanding human NM.     

The structure of the F-actin filament and the actin molecule.

A consensus view on the three-dimensional structure of the F-actin filament and the relative strength of the intersubunit contacts in the filament has been established from an atomic filament model and recent three-dimensional reconstructions from electron micrographs of F-actin filaments. Functional implications of recent structural and biochemical data indicating a rather dynamic filament structure are discussed.     

Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.     

A new muscle contractile system composed of a thick filament lattice and a single actin filament

To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.