Polymerization, three-dimensional structure and mechanical properties of Ddictyostelium versus rabbit muscle actin filaments

To assess more systematically functional differences among non-muscle and muscle actins and the effect of specific mutations on their function, we compared actin from Dictyostelium discoideum (D-actin) with actin from rabbit skeletal muscle (R-actin) with respect to the formation of filaments, their three-dimensional structure and mechanical properties. With Mg(2+) occupying the single high-affinity divalent cation-binding site, the course of polymerization is very similar for the two types of actin. In contrast, when Ca(2+ )is bound, D-actin exhibits a significantly longer lag phase at the onset of polymerization than R-actin. Crossover spacing and helical screw angle of negatively stained filaments are similar for D and R-F-actin filaments, irrespective of the tightly bound divalent cation. However, three-dimensional helical reconstructions reveal that the intersubunit contacts along the two long-pitch helical strands of D-(Ca)F-actin filaments are more tenuous compared to those in R-(Ca)F-actin filaments. D-(Mg)F-actin filaments on the other hand exhibit more massive contacts between the two long-pitch helical strands than R-(Mg)F-actin filaments. Moreover, in contrast to the structure of R-F-actin filaments which is not significantly modulated by the divalent cation, the intersubunit contacts both along and between the two long-pitch helical strands are weaker in D-(Ca)F-actin compared to D-(Mg)F-actin filaments. Consistent with these structural differences, D-(Ca)F-actin filaments were significantly more flexible than D-(Mg)F-actin.Taken together, this work documents that despite being highly conserved, muscle and non-muscle actins exhibit subtle differences in terms of their polymerization behavior, and the three-dimensional structure and mechanical properties of their F-actin filaments which, in turn, may account for their functional diversity.     

Polymerization and structure of nucleotide-free actin filaments

Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and approximately 99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA filaments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from filament ends. By electron microscopy of negatively stained specimens, NFA forms long filaments with a persistence length 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nm resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically significant less mass density between the two strands of NFA filaments. When compared with ADP-actin filaments, the major difference peak of NFA filaments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA filaments for rhodamine phalloidin is lower than that of native actin filaments, due to a lower association rate. This work confirms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of filaments through ATP hydrolysis and phosphate release, and to provide an internal timer for the age of the filament.     

Arrangement of the heads of myosin in relaxed thick filaments from tarantula muscle

Thick filaments from leg muscle of tarantula, maintained under relaxing conditions (Mg-ATP and EGTA), were negatively stained and photographed with minimal electron dose. Particles were selected for three-dimensional image reconstruction by general visual appearance and by the strength and symmetry of their optical diffraction patterns, the best of which extend to spacings of 1/5 nm-1. The helical symmetry is such that, on a given layer-line, Bessel function contributions of different orders start to overlap at fairly low resolution and must therefore be separated computationally by combining data from different views. Independent reconstructions agree well and show more detail than previous reconstructions of thick filaments from Limulus and scallop. The strongest feature is a set of four long-pitch right-handed helical ridges (pitch 4 X 43.5 nm) formed by the elongated myosin heads. The long-pitch helices are modulated to give ridges with an axial spacing of 14.5 nm, lying in planes roughly normal to the filament axis and running circumferentially. We suggest that the latter may be formed by the stacking of a subfragment 1 (S1) head from one myosin molecule on an S1 from an axially neighbouring molecule. Internal features in the map indicate an approximate local twofold axis relating the putative heads within a molecule. The heads appear to point in opposite directions along the filament axis and are located very close to the filament backbone. Thus, for the first time, the two heads of the myosin molecule appear to have been visualized in a native thick filament under relaxing conditions.     

Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (~75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.     

Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

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.     

Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction

Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.     

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)     

Dynamic and elastic properties of F-actin: a normal-modes analysis.

We examine the dynamic, elastic, and mechanical consequences of the proposed atomic models of F-actin, using a normal mode analysis. This initial analysis is done in vacuo and assumes that all monomers are rigid and equivalent. Our computation proceeds from the atomic level and, relying on a single fitting parameter, reproduces various experimental results, including persistence lengths, elastic moduli, and contact energies. The computations reveal modes of motion characteristic to all polymers, such as longitudinal pressure waves, torsional waves, and bending, as well as motions unique to F-actin. Motions typical to actin include a "groove-swinging" motion of the two long-pitch helices, as well as an axial slipping motion of the two strands. We prepare snapshots of thermally activated filaments and quantify the accumulation of azimuthal angular "disorder," variations in cross-over lengths, and various other fluctuations. We find that the orientation of a small number of select residues has a surprisingly large effect on the filament flexibility and elasticity characteristics.     

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.     

Probing the structure of F-actin: cross-links constrain atomic models and modify actin dynamics.

Cross-links between protomers in F-actin can be used as a very sensitive probe of both the dynamics and structure of F-actin. We have characterized filaments formed from a previously described yeast actin Q41C mutant, where disulfide bonds can be formed between the Cys41 that is introduced into subdomain-2 and Cys374 on an adjacent protomer. We find that the distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability cross-linking reaction. The random distribution suggests that disulfide formation does not cause a significant perturbation of the F-actin structure. Consistent with this lack of perturbation, three-dimensional reconstructions of extensively cross-linked filaments, using a new approach to helical image analysis, show very small structural changes with respect to uncross-linked filaments. This finding is in conflict with refined models but in agreement with the original Holmes et al. model for F-actin. Under conditions where 94 % of the protomers are linked by disulfide bonds, the distribution of filament twist becomes more heterogeneous with respect to control filaments. A molecular model suggests that strain, introduced by the disulfide, is relieved by increasing the twist of the long-pitch actin helices. Disulfide formation makes yeast actin filaments approximately three times less flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin. These observations support previous reports that the rigidity of F-actin can be controlled by the position of subdomain-2, and that this region is more flexible in yeast F-actin than in skeletal muscle F-actin.     

Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.     

Dolastatin 11 connects two long-pitch strands in F-actin to stabilize microfilaments

Dolastatin 11, a drug isolated from the Indian Ocean sea hare Dolabella auricularia, arrests cytokinesis in vivo and increases the amount of F-actin to stabilize F-actin in vitro, like phalloidin and jasplakinolide. However, according to the previous biochemical study, the binding of dolastatin 11 to F-actin does not compete with that of phalloidin, suggesting that the binding sites are different. To understand the mechanism of F-actin stabilization by dolastatin 11, we determined the position of bound dolastatin 11 in F-actin using the X-ray fiber diffraction from oriented filament sols. Our analysis shows that the position of dolastatin 11 is clearly different from that of phalloidin. However, these bound drugs are present in the gap between the two long-pitch F-actin strands in a similar way. The result suggests that the connection between the two long-pitch F-actin strands might be a key for the control of F-actin stabilization.     

AN ultrastructural study of cross-bridge arrangement in the frog thigh muscle thick filament.

We have developed thick filament isolation methods that preserve the relaxed cross-bridge order of frog thick filaments such that the filaments can be analyzed by the convergent techniques of electron microscopy, optical diffraction, and computer image analysis. Images of the filaments shadowed by using either unidirectional shadowing or rotary shadowing show a series of subunits arranged along a series of right-handed near-helical strands that occur every 43 nm axially along the filament arms. Optical filtrations of images of these shadowed filaments show 4-5 subunits per half-turn of the strands, consistent with a three-stranded arrangement of the cross-bridges, thus supporting our earlier results from negative staining and computer-image analysis. The optical diffraction patterns of the shadowed filaments show a departure from the pattern expected for helical symmetry consistent with the presence of cylindrical symmetry and a departure of the cross-bridges from helical symmetry. We also describe a modified negative staining procedure that gives improved delineation of the cross-bridge arrangement. From analysis of micrographs of these negatively stained filament tilted about their long axes, we have computed a preliminary three-dimensional reconstruction of the filament that clearly confirms the three-stranded arrangement of the myosin heads.     

Structure of actin filament bundles from microvilli of sea urchin eggs

The detailed substructure of actin filament bundles in microvilli of fertilized sea urchin eggs has been studied by analysing electron microscope images of negatively stained specimens. Transverse stripes which repeat about every 130 Å along the axis of a bundle, as previously observed by Burgess & Schroeder (1977), reflect the positions of cross-bridges that connect the filaments into a bundle. Analysis of optical transforms of the micrographs reveals that there are approximately 14 actin monomers between cross-overs of the two long-pitch helical strands of the actin filaments, with three cross-bridges in this interval. The structure is basically similar to that of the hexagonally packed bundles prepared in vitro from high speed supernatants of sea urchin eggs by Kane (1975) and analyzed by DeRosier et al. (1977). One clear difference, however, is that the in vivo microvillar filament bundles are supercoiled, giving rise to long axial repeats of 1500 to 2000 Å.Computationally filtered images of regions that were only slightly supercoiled reveal the relative alignment of filaments within the bundles and show that crossbridges appear to interact with four actin monomers, apparently linking two actin monomers on one strand of one filament to the nearest two monomers on a neighbouring filament. However, the cross-bridges are not spaced at equal intervals corresponding to four actin subunits, presumably because of the lack of hexagonal symmetry in the individual filaments, which have about 14 actin monomers between cross-overs. Instead, the cross-bridges are arranged quasiequivalently along the longitudinal axis of the bundles, in steps of four or five actin subunit spacings (28 Å each).     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

A molecular switch: subunit rotations involved in the right-handed to left-handed transitions of Salmonella typhimurium flagellar filaments

Using the combined techniques of cryoelectron microscopy and image analysis, we generated three-dimensional reconstructions of flagellar filaments from straight, right-handed (SJW1655-R) and straight, left-handed (SJW1660-L) Salmonella typhimurium mutants, both of which have the same parental strain (SJW1103). In the filaments from SJW1655, all flagellin subunits have the same conformation (R), while in filaments from SJW1660, the subunits are all in the alternate (L) conformation. The difference between the two three-dimensional density maps reveal the structural changes that accompany switching of the flagellin subunits between the two conformations. In going from the R to L state, the subunit undergoes a rotation 30 degrees clockwise about a radial axis and 38 degrees clockwise about a vertical axis, and suffers a 50 degrees bend of the outer, relative to the inner, subunit domain. The intersubunit spacing, along the 11-start protofilaments, changes from 51.6 A in the right-handed filament to 52.1 A in the left-handed filament. In order to produce the correct corkscrew shape in native filaments, the change in contacts that produces this shortening of 0.5 A must occur among the inner domains at a radius of about 30 A. We suggest that the changes in the middle domains of the subunit are the switch that forces changes in the inner domains.     

Conformational switching in the flagellar filament of Salmonella typhimurium.

The flagellar filament of the mutant Salmonella typhimurium strain SJW814 is straight, and has a right-handed twist like the filament of SJW1655. Three-dimensional reconstructions from electron micrographs of ice-embedded filaments reveal a flagellin subunit that has the same domain organization as that of SJW1655. Both show slight changes from the domain organization of the subunits from SJW1660, which possesses a straight, left-handed filament. This points to the possible role of changes in subunit conformation in the left-to-right-handed structural transition in filaments. Comparison of the left and right-handed filaments shows that the subunit's orientation and intersubunit bonding appear to change. The orientation of the subunit in the SJW814 filament is intermediate between that of SJW1655 and SJW1660. Its intermediate orientation may explain why the filaments of SJW1655 and SJW1660 are locked in one conformation, whereas the filament of SJW814 can be induced to switch by, for example, changes in pH and ionic strength.     

Helical reconstruction of frozen-hydrated scallop myosin filaments.

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.