Cryo-atomic force microscopy of smooth muscle myosin.

The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low probability in the paircoil score. The second major bend was 100 nm from the H-T junction in nonphosphorylated and closer to a skip residue than the bend (at 95 nm) in thiophosphorylated molecules. The shorter tail and distance between the two major bends induced by thiophosphorylation are interpreted to result from melting of the coiled-coil. An additional bend not previously reported occurred, with a lower frequency, approximately 24 nm from the H-T. The range of separation between the two heads was greater in thiophosphorylated molecules. Occasional high-resolution images showed slight unwinding of the coiled-coil of the base of the heads. We suggest that phosphorylation of MLC20 can affect the structure of extended, 6S myosin.     

Skip residues correlate with bends in the myosin tail

Sharp bends have previously been observed in the tail of the skeletal myosin molecule at well-defined positions 44, 75 and 135 nm from the head-tail junction, and in vertebrate smooth myosin at two positions about 45 and 96 nm from this junction. The amino acid sequence of the heavy chain does not straightforwardly account for such bending on the original model of the tail in which an invariant proline residue is present at the head-tail junction and the repeating seven amino acid pattern of hydrophobic residues lies entirely in the tail. Recently, a revised model has been proposed by Rimm et al. in which the first seven to eight heptads lie in the heads. It is shown here that with this model the observed bends in the tail of skeletal myosin coincide with three of the four additional (skip) residues that interrupt the heptad repeat. It is concluded that the skip residues, by causing localized instability of the coiled-coil, are responsible for the bends. Smooth myosin lacks the second of these skip residues explaining the absence of a bend at 75 nm.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

The tail domain of myosin Va modulates actin binding to one head

Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.     

3D Structure of fish muscle myosin filaments

Myosin filaments isolated from goldfish (Carassius auratus) muscle under relaxing conditions and viewed in negative stain by electron microscopy have been subjected to 3D helical reconstruction to provide details of the myosin head arrangement in relaxed muscle. Previous X-ray diffraction studies of fish muscle (plaice) myosin filaments have suggested that the heads project a long way from the filament surface rather than lying down flat and that heads in a single myosin molecule tend to interact with each other rather than with heads from adjacent molecules. Evidence has also been presented that the head tilt is away from the M-band. Here we seek to confirm these conclusions using a totally independent method. By using 3D helical reconstruction of isolated myosin filaments the known perturbation of the head array in vertebrate muscles was inevitably averaged out. The 3D reconstruction was therefore compared with the X-ray model after it too had been helically averaged. The resulting images showed the same characteristic features: heads projecting out from the filament backbone to high radius and the motor domains at higher radius and further away from the M-band than the light-chain-binding neck domains (lever arms) of the heads.     

Two-headed binding of the unphosphorylated nonmuscle heavy meromyosin.ADP complex to actin

The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA heavy meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated heavy meromyosin bind very strongly to actin (K(d) < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (K(d) around 1 microM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myosin IIA subfragment-1 (Kovács et al. (2003) J. Biol. Chem. 278, 38132.). Furthermore, they imply that the structure of the two heads in the ternary actomyosin-ADP complex is symmetrical and that the asymmetrical structure observed in the presence of ATP and the absence of actin in previous investigations (Wendt et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361) is likely to represent an ATPase intermediate that precedes the actomyosin-ADP state.     

Shape and flexibility of the myosin molecule

Molecules of rabbit skeletal myosin have been examined in the electron microscope after drying at low temperature from solutions containing ethylene glycol or glycerol and rotary-shadowing with platinum. Analysis of the structure has been assisted by stereo-photography. While the general appearance, two heads attached to a long tail, is similar to that described by Slayter & Lowey (1967), more detail about the shape and size of the heads can be discerned and new information has been obtained about the flexibility of the tail and the head-tail junction.The heads are 190 Å long and wider at their ends than near the junction with the tail; the shape resembles that of a pear. The length is appreciably greater than the generally accepted value for subfragment 1, the proteolytic fragment of myosin. The heads are flexibly attached to the tail and can assume a wide range of tilt angles.Because the point where the two heads join the tail can be identified, the length of the tail, 1560 (±50) Å, can be measured more accurately than formerly. While all parts of the tail are somewhat flexible, sharp bends often occur at a well-defined site 430 Å from the head-tail junction. The demonstration of hinges at the head-tail junction and in the tail provides strong support for H. E. Huxley's (1969) hypothesis for the mechanism of muscle contraction.     

Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction

The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.     

Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.     

Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.     

Flexibility within Myosin Heads Revealed by Negative Stain and Single-Particle Analysis

Electron microscopy of negatively stained myosin has previously revealed three discrete regions within the heads of the molecule. However, despite a probable resolution of ∼2 nm, it is difficult to discern directly consistent details within these regions. This is due to variability in both head conformation and in staining. In this study, we applied single-particle image processing and classified heads into homogeneous groups. The improved signal-to-noise ratio after averaging these groups reveals substantially improved detail. The image averages were compared to a model simulating negative staining of the atomic structure of subfragment-1 (S1). This shows that the three head regions correspond to the motor domain and the essential and regulatory light chains. The image averages were very similar to particular views of the S1 model. They also revealed considerable flexibility between the motor and regulatory domains, despite the molecules having been prepared in the absence of nucleotide. This flexibility probably results from rotation of the regulatory domain about the motor domain, where the relative movement of the regulatory light chain is up to 12 nm, and is most clearly illustrated in animated sequences (available at http://www.leeds.ac.uk/chb/muscle/ myosinhead.html). The sharply curved conformation of the atomic model of S1 is seen only rarely in our data, with straighter heads being more typical.Muscle force is derived from the interaction of myosin and actin. The most likely mechanisms involve a large conformational change in the attached head of the myosin molecule, either in the binding angle made with actin (Huxley, 1969), or within the head itself (Rayment et al., 1993a ). Attached heads may also deform elastically in response to stress, allowing storage of strain energy derived from ATP or stretching. Information about the structure and flexibility of the myosin head is therefore important in exploring the molecular mechanism of force generation.The most detailed electron micrographs of myosin molecules have been obtained by negative staining and have revealed three discrete regions within the heads. The most common appearance consists of a large distal region connected to the head–tail junction by two smaller regions in line (Walker et al., 1985; Walker and Trinick, 1986, 1988). After the subsequent elucidation of the atomic structure of myosin subfragment-1 (S1)¹ by X-ray crystallography (Rayment et al., 1993b), it seemed likely that the three regions identified correspond to the motor domain and the essential and regulatory light chains. However, although the micrographs show many details, probably to a resolution of ∼2 nm, further analysis is hindered by the wide variations in head appearance and by the considerable noise caused by the size and variability of the stain crystallites.It is possible to recover more information from such micrographs by using the image processing technique known as “single-particle analysis” (Frank, 1996). This allows images with similar appearances to be classified into homogeneous groups; these can then be averaged, thus improving the signal-to-noise ratio. This type of approach was used previously with the heads of shadowed myosin molecules (Vibert, 1988). Here we describe its application to negatively stained molecules, which reveals a considerable amount of new detail. To confirm the validity of our results we developed a negative stain model and simulated staining of the atomic structure of S1. This showed that the three regions in stained myosin heads are indeed the motor domain and the essential and regulatory light chains (the regulatory domain).Initial attempts to classify the images based on the appearances of whole heads were difficult to interpret. It emerged that heads not only adopt a variety of orientations on the grid, but also a variety of conformations between the motor and regulatory domains. Single particle analysis has been used previously, mainly with invariant structures. Therefore, we classified heads twice: first using only the motor domains, and then using only the regulatory domains. The data from both rounds of classification were then combined. The results demonstrate a large degree of flexibility within heads.     

Negative staining of myosin molecules

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.     

Calcium and cargoes as regulators of myosin 5a activity

Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.     

The Kinesin-1 Tail Conformationally Restricts the Nucleotide Pocket

We have used electron paramagnetic resonance and fluorescence spectroscopy to study the interaction between the kinesin-1 head and its regulatory tail domain. The interaction between the tails and the enzymatically active heads has been shown to inhibit intrinsic and microtubule-stimulated ADP release. Here, we demonstrate that the probe mobility of two different spin-labeled nucleotide analogs in the kinesin-1 nucleotide pocket is restricted upon binding of the tail domain to kinesin-1 heads. This conformational restriction is distinct from the microtubule-induced changes in the nucleotide pocket. Unlike myosin V, this tail-induced restriction occurs independent of nucleotide state. We find that the head-tail interaction that causes the restriction only weakly stabilizes Mg²⁺ in the nucleotide pocket. The conformational restriction also occurs when a tail construct containing a K922A point mutation is used. This mutation eliminates the tail's ability to inhibit ADP release, indicating that the tail does not inhibit nucleotide ejection from the pocket by simple steric hindrance. Together, our data suggest that the observed head-tail interaction serves as a scaffold to position K922 to exert its inhibitory effect, possibly by interacting with the nucleotide α/β-phosphates in a manner analogous to the arginine finger regulators of some G proteins.     

Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail

Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope studies and the peptide mapping and competitive binding studies we have established the binding sites of 16 of 23 monoclonal antibodies. Two of the 23 antibodies block the formation of myosin-II filaments and given sufficient time, disassemble preformed myosin-II filaments. Both antibodies bind near one another at the tip of the myosin-II tail and are those that decorate the bare zone of preformed bipolar filaments with 14-nm periodicity. None of the other antibodies affect myosin filament formation, including one that binds to another site near the tip of the myosin-II tail. This demonstrates that antibodies can inhibit polymerization of myosin-II, but only when they bind to key sites on the tail of the molecule.     

Location of the head-tail junction of myosin

The tails of double-headed myosin molecules consist of an alpha-helical/coiled-coil structure composed of two identical polypeptides with a heptad repeat of hydrophobic amino acids that starts immediately after a conserved proline near position 847. Both muscle and nonmuscle myosins have this heptad repeat and it has been assumed that proline 847 is physically located at the head-tail junction. We present two lines of evidence that this assumption is incorrect. First, we localized the binding sites of several monoclonal antibodies on Acanthamoeba myosin-II both physically, by electron microscopy, and chemically, with a series of truncated myosin-II peptides produced in bacteria. These data indicate that the head-tail junction is located near residue 900. Second, we compared the lengths of two truncated recombinant myosin-II tails with native myosin-II. The distances from the NH2 termini to the tips of these short tails confirms the rise per residue (0.148 nm/residue) and establishes that the 86-nm tail of myosin-II must start near residue 900. We propose that the first 53 residues of heptad repeat of Acanthamoeba myosin-II and other myosins are located in the heads and the proteolytic separation of S-1 from rod occurs within the heads.     

Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation

Dictyostelium myosin has been examined under conditions that reveal intramolecular and intermolecular interactions that may be important in the process of assembly and its regulation. Rotary shadowed myosin molecules exhibit primarily two configurations under these conditions: straight parallel dimers and folded monomers. All of the monomers bend in a specific region of the 1860-A-long tail that is 1200 A from the head-tail junction. Molecules in parallel dimers are staggered by 140 A, which is a periodicity in the packing of myosin molecules originally observed in native thick filaments of muscle. The most common region for interaction in the dimers is a segment of the tail about 200-A-long, extending from 900 to 1100 A from the head-tail junction. Parallel dimers form tetramers by way of antiparallel interactions in their tail regions with overlaps in multiples of 140 A. The folded configuration of the myosin molecules is promoted by phosphorylation of the heavy chain by Dictyostelium myosin heavy chain kinase. It appears that the bent monomers are excluded from filaments formed upon addition of salt while the dimeric molecules assemble. These results may provide the structural basis for primary steps in myosin filament assembly and its regulation by heavy chain phosphorylation.     

Head-head interaction characterizes the relaxed state of Limulus muscle myosin filaments

Regulation of muscle contraction via the myosin filaments occurs in vertebrate smooth and many invertebrate striated muscles. Studies of unphosphorylated vertebrate smooth muscle myosin suggest that activity is switched off through an intramolecular interaction between the actin-binding region of one head and the converter and essential light chains of the other, inhibiting ATPase activity and actin interaction. The same interaction (and additional interaction with the tail) is seen in three-dimensional reconstructions of relaxed, native myosin filaments from tarantula striated muscle, suggesting that such interactions are likely to underlie the off-state of myosin across a wide spectrum of the animal kingdom. We have tested this hypothesis by carrying out cryo-electron microscopy and three-dimensional image reconstruction of myosin filaments from horseshoe crab (Limulus) muscle. The same head-head and head-tail interactions seen in tarantula are also seen in Limulus, supporting the hypothesis. Other data suggest that this motif may underlie the relaxed state of myosin II in all species (including myosin II in nonmuscle cells), with the possible exception of insect flight muscle.The molecular organization of the myosin tails in the backbone of muscle thick filaments is unknown and may differ between species. X-ray diffraction data support a general model for crustaceans in which tails associate together to form 4-nm-diameter subfilaments, with these subfilaments assembling together to form the backbone. This model is supported by direct observation of 4-nm-diameter elongated strands in the tarantula reconstruction, suggesting that it might be a general structure across the arthropods. We observe a similar backbone organization in the Limulus reconstruction, supporting the general existence of such subfilaments.     

Probing Muscle Myosin Motor Action: X-Ray (M3 and M6) Interference Measurements Report Motor Domain Not Lever Arm Movement

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.     

Refined structure of bony fish muscle myosin filaments from low-angle X-ray diffraction data

Application of X-ray diffraction methods to the elucidation of the arrangement of the myosin heads on myosin filaments in resting muscles is made simpler when the muscles themselves are well ordered in 3D. Bony fish muscle for the vertebrates and insect flight muscle for the invertebrates are the muscles of choice for this analysis. The rich, well-sampled, low-angle X-ray diffraction pattern from bony fish muscle has previously been modelled with an R-factor of 3.4% between observed and calculated transforms on the assumption that the two heads in one myosin molecule have the same shape. However, recent evidence from other kinds of analysis of other muscles has shown that this assumption may not be valid. There is evidence that the motor domain of one head in each pair may interact with the neck region of the second head. This possibility has been tested directly in the present analysis which extends the X-ray modelling of fish muscle myosin filaments by permitting independent shape changes of the two heads in one molecule. The new model, with a computed R-factor of 1.19% against 56 independent reflections, shows that in fish muscle also there is a marked asymmetry in the organisation of each head pair.