Thin filament flexibility and its role in muscle contraction

Contemporary experimental methods do not allow unequivocal determination of molecular structural events during muscle contraction. To analyze existing contradictions, an original computer program has been developed. This program reconstructs the hexagonal lattice of a sarcomere for different states of muscle and finds the most realistic structure by comparing the calculated Fourier spectrum with the actual diffraction pattern. Previously, the new approach allowed reconstructing the actual structure of a myosin filament from mammalian striated muscle (http://zope.ibib.waw.pl/pspk). In this work, the thin filament is reconstructed for three states: relaxed, activated, and contracting. The good fit between the calculated Fourier spectra and the actual diffraction patterns taken from the literature suggests that the thin filament owing to its flexibility may play an active role in muscle contraction, as myosin cross-bridges do.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy.

Rapid freezing followed by freeze-substitution has been used to study the ultrastructure of the myosin filaments of live and demembranated frog sartorius muscle in the states of relaxation and rigor. Electron microscopy of longitudinal sections of relaxed specimens showed greatly improved preservation of thick filament ultrastructure compared with conventional fixation. This was revealed by the appearance of a clear helical arrangement of myosin crossbridges along the filament surface and by a series of layer line reflections in computed Fourier transforms of sections, corresponding to the layer lines indexing on a 43 nm repeat in X-ray diffraction patterns of whole, living muscles. Filtered images of single myosin filaments were similar to those of negatively stained, isolated vertebrate filaments and consistent with a three-start helix. M-line and other non-myosin proteins were also very well preserved. Rigor specimens showed, in the region of overlapping myosin and actin filaments, periodicities corresponding to the 36, 24, 14.4 and 5.9 nm repeats detected in X-ray patterns of whole muscle in rigor; in the H-zone they showed a disordered array of crossbridges. Transverse sections, whose Fourier transforms extend to the (3, 0) reflection, supported the view, based on X-ray diffraction and conventional electron microscopy, that in the overlap zone of relaxed muscle most of the crossbridges are detached from the thin filaments while in rigor they are attached. We conclude that the rapid freezing technique preserves the molecular structure of the myofilaments closer to the in vivo state (as monitored by X-ray diffraction) than does normal fixation.     

Calcium regulates scallop muscle by changing myosin flexibility

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called “thick filament regulation” is quite different to vertebrate striated muscle which is switched on and off via “thin filament regulation” whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation. This paper is not being submitted elsewhere and the authors have no competing financial interests     

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, “free” and “blocked”, formed an asymmetric structure named the “interacting-heads motif” (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca²⁺-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.     

Direct modeling of X-ray diffraction pattern from contracting skeletal muscle

A direct modeling approach was used to quantitatively interpret the two-dimensional x-ray diffraction patterns obtained from contracting mammalian skeletal muscle. The dependence of the calculated layer line intensities on the number of myosin heads bound to the thin filaments, on the conformation of these heads and on their mode of attachment to actin, was studied systematically. Results of modeling are compared to experimental data collected from permeabilized fibers from rabbit skeletal muscle contracting at 5°C and 30°C and developing low and high isometric tension, respectively. The results of the modeling show that: i), the intensity of the first actin layer line is independent of the tilt of the light chain domains of myosin heads and can be used as a measure of the fraction of myosin heads stereospecifically attached to actin; ii), during isometric contraction at near physiological temperature, the fraction of these heads is ∼40% and the light chain domains of the majority of them are more perpendicular to the filament axis than in rigor; and iii), at low temperature, when isometric tension is low, a majority of the attached myosin heads are bound to actin nonstereospecifically whereas at high temperature and tension they are bound stereospecifically.     

Packing of α-Helical Coiled-Coil Myosin Rods in Vertebrate Muscle Thick Filaments

Muscle myosin filament backbones are aggregates of long coiled-coil α-helical myosin rods, with the myosin heads arranged approximately helically on the filament surface, but the details of the rod packing are not known. Computed Fourier transforms of plausible molecular packing models for the vertebrate striated muscle myosin filament have been compared with observed high-angle X-ray diffraction patterns from plaice fin muscle. Models considered include those in which the coiled-coil rod parts of myosin are packed into various kinds of subfilaments or into a curved molecular crystalline layer. A general conclusion is that if the myosin rods are tilted by less than about 1° or more than about 3° from the filament long axis, very poor agreement is obtained between the computed and observed high-angle diffraction patterns. Qualitative comparison of calculated Fourier transforms, taken together with electron micrograph information, shows that the curved molecular crystal model and a model with hexagonally close-packed 4-nm subfilaments appear to explain the whole set of observations more satisfactorily than the alternatives. It is argued on other grounds that of these two possibilities the curved molecular crystal model is the more plausible.     

Effects of a cardiomyopathy-causing troponin t mutation on thin filament function and structure

Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the thin filament or thick filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated thin filament target, troponin T. Electron microscope observations showed that the thin filament undergoes normal structural changes in response to Ca(2+) binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the thin filament.     

The vertebrate skeletal muscle thick filaments are not three-stranded. Reinterpretation of some experimental data

Computer simulation of mass distribution within the model and Fourier transforms of images depicting mass distribution are explored for verification of two alternative modes of the myosin molecule arrangement within the vertebrate skeletal muscle thick filaments. The model well depicting the complete bipolar structure of the thick filament and revealing a true threefold-rotational symmetry is a tube covered by two helices with a pitch of 2 x 43 nm due to arrangement of the myosin tails along a helical path and grouping of all myosin heads in the crowns rotated by 240 degrees and each containing three cross-bridges separated by 0 degrees, 120 degrees, and 180 degrees. The cross-bridge crown parameters are verified by EM images as well as by optical and low-angle X-ray diffraction patterns found in the literature. The myosin tail arrangement, at which the C-terminus of about 43-nm length is near-parallel to the filament axis and the rest of the tail is quite strongly twisted around, is verified by the high-angle X-ray diffraction patterns. A consequence of the new packing is a new way of movement of the myosin cross-bridges, namely, not by bending in the hinge domains, but by unwrapping from the thick filament surface towards the thin filaments along a helical path.     

Structural transients of contractile proteins upon sudden ATP liberation in skeletal muscle fibers

Structural changes of contractile proteins were examined by millisecond time-resolved two-dimensional x-ray diffraction recordings during relaxation of skinned skeletal muscle fibers from rigor after caged ATP photolysis. It is known that the initial dissociation of the rigor actomyosin complex is followed by a period of transient active contraction, which is markedly prolonged in the presence of ADP by a mechanism yet to be clarified. Both single-headed (overstretched muscle fibers with exogenous myosin subfragment-1) and two-headed (fibers with full filament overlap) preparations were used. Analyses of various actin-based layer line reflections from both specimens showed the following: 1), The dissociation of the rigor actomyosin complex was fast and only modestly decelerated by ADP and occurred in a single exponential manner without passing through any detectable transitory state. Its ADP sensitivity was greater in the two-headed preparation but fell short of explaining the large ADP effect on the transient active contraction. 2), The decay of the activated state of the thin filament followed the time course of tension more closely in an ADP-dependent manner. These results suggest that the interplay between the reattached active myosin heads and the thin filament is responsible for the prolonged active contraction in the presence of ADP.     

Resolution of three structural states of spin-labeled myosin in contracting muscle.

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.     

States of thin filament regulatory proteins as revealed by combined cross-linking/X-ray diffraction techniques

The regulatory protein system in the skeletal muscle thin filaments is known to exhibit three discrete states, called "off" or "blocked" (no Ca2+), "on" or "closed" (with Ca2+ alone) and "potentiated" or "open" (with strongly bound myosin head) states. Biochemical studies have shown that only weak interactions with myosin are allowed in the second state. Characterization of each state is often difficult, because the equilibria among these states are readily shifted by experimental conditions. To overcome this problem, we chemically cross-linked the skeletal muscle thin filament in the three states with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), in overstretched muscle fibers. The state of the regulatory proteins was monitored by measuring the intensity of the second actin layer-line (2nd LL) reflection in X-ray diffraction patterns. Structurally, the thin filaments cross-linked in the three states exhibited three corresponding discrete levels of 2nd LL intensities, which were not Ca2+-sensitive any more. Functionally, the thin filament cross-linked in the "off-blocked" state inhibited strong interaction with myosin head (subgfragment-1 or S1). The thin filament cross-linked in the "potentiated-open" state allowed strong interaction and full ATPase activity of S1 as described previously. The thin filament cross-linked in the "on-closed" state allowed strong interactions with S1 and actin-activated ATPase without enhancing the 2nd LL to the level of "potentiated-open" state, contrary to the expectations from the biochemical studies. The results demonstrate the potential of EDC as a tool for studying the states of calcium regulation, and the apparent uncoupling between the 2nd LL intensity and the function provides a new insight into the mechanism of thin filament regulation.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

"Crystalline" myosin cross-bridge array in relaxed bony fish muscle. Low-angle x-ray diffraction from plaice fin muscle and its interpretation

Detailed structural analysis of muscles normally used to study myosin cross-bridge behavior (e.g., frog sartorius muscle, insect flight muscle) is extremely difficult due to the statistical disorder inherent in their myosin filament arrays. Bony fish muscle is different from all other muscle types in having a myosin filament (A-Band) array with good three-dimensional (crystalline) regularity that is coherent right across each myofibril. Rigorous structure analysis is feasible with fish muscle. We show that low-angle x-ray diffraction patterns from plaice fin muscle contain characteristic vertebrate layer lines at orders of 429 (+/- 0.2) A, that these layer lines are well sampled by row-lines from a simple hexagonal lattice of a-spacing 470 (+/- 2.0) A at rest length and that there are meridional reflections, due to axial perturbations of the basic helix of myosin heads, similar in position to those from frog muscle but differing in relative intensities. Clear trends based on modeling to a resolution of 130 A of the observed intensities in the low angle x-ray diffraction pattern from relaxed plaice fin muscle suggest that: (a) the pattern out to 130 A is more sensitive to the distribution of the two heads than it is to details of the head shape, (b) both heads in one myosin molecule probably tilt axially in the same direction by approximately 20-40 degrees relative to a normal to the thick filament backbone, (c) the center of mass of the heads is at 145 to 160 A radius, and (d) the two heads form a compact structure by lying closely adjacent to each other and almost parallel. Little rotational disorder of the heads can occur. Because of its crystallinity, bony fish muscle provides a uniquely useful structural probe of myosin cross-bridge behavior in other muscle states such as rigor and active contraction.     

Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments

Tropomyosin movements on thin filaments are thought to sterically regulate muscle contraction, but have not been visualized during active filament sliding. In addition, although 3-D visualization of myosin crossbridges has been possible in rigor, it has been difficult for thick filaments actively interacting with thin filaments. In the current study, using three-dimensional reconstruction of electron micrographs of interacting filaments, we have been able to resolve not only tropomyosin, but also the docking sites for weak and strongly bound crossbridges on thin filaments. In relaxing conditions, tropomyosin was observed on the outer domain of actin, and thin filament interactions with thick filaments were rare. In contracting conditions, tropomyosin had moved to the inner domain of actin, and extra density, reflecting weakly bound, cycling myosin heads, was also detected, on the extreme periphery of actin. In rigor conditions, tropomyosin had moved further on to the inner domain of actin, and strongly bound myosin heads were now observed over the junction of the inner and outer domains. We conclude (1) that tropomyosin movements consistent with the steric model of muscle contraction occur in interacting thick and thin filaments, (2) that myosin-induced movement of tropomyosin in activated filaments requires strongly bound crossbridges, and (3) that crossbridges are bound to the periphery of actin, at a site distinct from the strong myosin binding site, at an early stage of the crossbridge cycle.     

Hugh E. Huxley: The Compleat Biophysicist

The sliding filament model of muscle contraction, put forward by Hugh Huxley and Jean Hanson in 1954, is 60 years old in 2014. Formulation of the model and subsequent proof was driven by the pioneering work of Hugh Huxley (1924–2013). We celebrate Huxley’s integrative approach to the study of muscle contraction; how he persevered throughout his career, to the end of his life at 89 years, to understand at the molecular level how muscle contracts and develops force. Here we show how his life and work, with its focus on a single scientific problem, had impact far beyond the field of muscle contraction to the benefit of multiple fields of cellular and structural biology. Huxley introduced the use of x-ray diffraction to study the contraction in living striated muscle, taking advantage of the paracrystalline lattice that would ultimately allow understanding contraction in terms of single molecules. Progress required design of instrumentation with ever-increasing spatial and temporal resolution, providing the impetus for the development of synchrotron facilities used for most protein crystallography and muscle studies today. From the time of his early work, Huxley combined electron microscopy and biochemistry to understand and interpret the changes in x-ray patterns. He developed improved electron-microscopy techniques, thin sections and negative staining, that enabled answering major questions relating to the structure and organization of thick and thin filaments in muscle and the interaction of myosin with actin and its regulation. Huxley established that the ATPase domain of myosin forms the crossbridges of thick filaments that bind actin, and introduced the idea that myosin makes discrete steps on actin. These concepts form the underpinning of cellular motility, in particular the study of how myosin, kinesin, and dynein motors move on their actin and tubulin tracks, making Huxley a founder of the field of cellular motility.     

Hugh E. Huxley: The Compleat Biophysicist

The sliding filament model of muscle contraction, put forward by Hugh Huxley and Jean Hanson in 1954, is 60 years old in 2014. Formulation of the model and subsequent proof was driven by the pioneering work of Hugh Huxley (1924–2013). We celebrate Huxley’s integrative approach to the study of muscle contraction; how he persevered throughout his career, to the end of his life at 89 years, to understand at the molecular level how muscle contracts and develops force. Here we show how his life and work, with its focus on a single scientific problem, had impact far beyond the field of muscle contraction to the benefit of multiple fields of cellular and structural biology. Huxley introduced the use of x-ray diffraction to study the contraction in living striated muscle, taking advantage of the paracrystalline lattice that would ultimately allow understanding contraction in terms of single molecules. Progress required design of instrumentation with ever-increasing spatial and temporal resolution, providing the impetus for the development of synchrotron facilities used for most protein crystallography and muscle studies today. From the time of his early work, Huxley combined electron microscopy and biochemistry to understand and interpret the changes in x-ray patterns. He developed improved electron-microscopy techniques, thin sections and negative staining, that enabled answering major questions relating to the structure and organization of thick and thin filaments in muscle and the interaction of myosin with actin and its regulation. Huxley established that the ATPase domain of myosin forms the crossbridges of thick filaments that bind actin, and introduced the idea that myosin makes discrete steps on actin. These concepts form the underpinning of cellular motility, in particular the study of how myosin, kinesin, and dynein motors move on their actin and tubulin tracks, making Huxley a founder of the field of cellular motility.     

Structural proteins in the myofilaments and regulation of contraction in vertebrate smooth muscle.

The contractile systems of vertebrate smooth and striated muscles are compared. Smooth muscles contain relatively large amounts of actin and tropomyosin organized into thin filaments, and smaller amounts of myosin in the form of thick filaments. The protein contents are consistent with observed thin:thick filament ratios of about 15-18:1 in smooth compared to 2:1 in striated muscle. The basic characteristics of both types of contractile proteins are similar; but there are a variety of quantitative differences in protein structures, enzymatic activities and filament stabilities. Biochemical and X-ray diffraction data generally support recent ultrastructural evidence concerning the organization of the myofilaments in smooth muscle, although a basic contractile unit comparable to the sarcomere in striated muscle has not been discerned. Myofilament interactions and contraction in smooth muscle are controlled by changes in the Ca2+ concentration. Recent evidence suggests the Ca2+-binding regulatory site is associated with the myosin in vertebrate smooth muscle (as in a variety of invertebrate muscles), rather than with troponin which is the regulatory protein associated with the thin filament in vertebrate striated muscle.     

A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle

The regulation of striated muscle contraction involves changes in the interactions of troponin and tropomyosin with actin thin filaments. In resting muscle, myosin-binding sites on actin are thought to be blocked by the coiled-coil protein tropomyosin. During muscle activation, Ca2+ binding to troponin alters the tropomyosin position on actin, resulting in cyclic actin-myosin interactions that accompany muscle contraction. Evidence for this steric regulation by troponin-tropomyosin comes from X-ray data [Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actin-containing filaments of verterbrate striated muscle. Cold Spring Habor Symp. Quant. Biol. 37, 341-352; Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Habor Symp. Quant. Biol. 37, 361-376; Parry, D.A., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33-55] and electron microscope (EM) data [Spudich, J.A., Huxley, H.E., Finch, J., 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632; O'Brien, E.J., Gillis, J.M., Couch, J., 1975. Symmetry and molecular arrangement in paracrystals of reconstituted muscle thin filaments. J. Mol. Biol. 99, 461-475; Lehman, W., Craig, R., Vibert, P., 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65-67] each with its own particular strengths and limitations. Here we bring together some of the latest information from EM analysis of single thin filaments from Pirani et al. [Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L.S., Lehman, W. (2005). Single particle analysis of relaxed and activated muscle thin filaments. J. Mol. Biol. 346, 761-772], with synchrotron X-ray data from non-overlapped muscle fibres to refine the models of the striated muscle thin filament. This was done by incorporating current atomic-resolution structures of actin, tropomyosin, troponin and myosin subfragment-1. Fitting these atomic coordinates to EM reconstructions, we present atomic models of the thin filament that are entirely consistent with a steric regulatory mechanism. Furthermore, fitting the atomic models against diffraction data from skinned muscle fibres, stretched to non-overlap to preclude crossbridge binding, produced very similar results, including a large Ca2+-induced shift in tropomyosin azimuthal location but little change in the actin structure or apparent alteration in troponin position.     

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+.

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.