Movement Of Tropomyosin Research Articles

Myosin's powerstroke transitions define atomic scale movement of cardiac thin filament tropomyosin.

Dynamic interactions between the myosin motor head on thick filaments and the actin molecular track on thin filaments drive the myosin-crossbridge cycle that powers muscle contraction. The process is initiated by Ca2+ and the opening of troponin-tropomyosin-blocked myosin-binding sites on actin. The ensuing recruitment of myosin heads and their transformation from pre-powerstroke to post-powerstroke conformation on actin produce the force required for contraction. Cryo-EM-based atomic models confirm that during this process, tropomyosin occupies three different average positions on actin. Tropomyosin pivoting on actin away from a TnI-imposed myosin-blocking position accounts for part of the Ca2+ activation observed. However, the structure of tropomyosin on thin filaments that follows pre-powerstroke myosin binding and its translocation during myosin's pre-powerstroke to post-powerstroke transition remains unresolved. Here, we approach this transition computationally in silico. We used the myosin helix-loop-helix motif as an anchor to dock models of pre-powerstroke cardiac myosin to the cleft between neighboring actin subunits along cardiac thin filaments. We then performed targeted molecular dynamics simulations of the transition between pre- and post-powerstroke conformations on actin in the presence of cardiac troponin-tropomyosin. These simulations show Arg 369 and Glu 370 on the tip of myosin Loop-4 encountering identically charged residues on tropomyosin. The charge repulsion between residues causes tropomyosin translocation across actin, thus accounting for the final regulatory step in the activation of the thin filament, and, in turn, facilitating myosin movement along the filament. We suggest that during muscle activity, myosin-induced tropomyosin movement is likely to result in unencumbered myosin head interactions on actin at low-energy cost.

Pivoting to a new view of tropomyosin movement.

JGP study (Lehman and Rynkiewicz. 2023. J. Gen. Physiol.https://doi.org/10.1085/jgp.202313387) suggests that tropomyosin regulates the crossbridge cycle in muscle by pivoting around relatively fixed points on actin thin filaments.

Myosin off-on transition modulates the simple harmonic motion of tropomyosin coupling length-dependent activation and the actomyosin chemomechanical cycle

Striated muscle contraction is modulated by structural changes in both myosin thick filaments and actin thin filaments. Yet, the signaling pathway between myofilaments is unknown. The thick filament mechanism involves movement of myosin heads (likely force/strain-dependent) from a folded, horizontal position on thick filament backbone (OFF) to a more perpendicular orientation (ON), closer to thin filament. Upon binding of calcium by troponin, the thin filament mechanism involves movement of tropomyosin from periphery of thin filament to the actin groove, enabling myosin crossbridge formation and force generation.

Myofilament glycation in diabetes reduces contractility by inhibiting tropomyosin movement, is rescued by cMyBPC domains

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 μM MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MG's functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosin's blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosin's position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF.

A Proposed Mechanism for the Initial Myosin Binding Event on the Cardiac Thin Filament: A Metadynamics Study.

The movement of tropomyosin over filamentous actin regulates the cross-bridge cycle of the thick with thin filament of cardiac muscle by blocking and revealing myosin binding sites. Tropomyosin exists in three, distinct equilibrium states with one state blocking myosin-actin interactions (blocked position) and the remaining two allowing for weak (closed position) and strong myosin binding (open position). However, experimental information illuminating how myosin binds to the thin filament and influences tropomyosin's transition across the actin surface is lacking. Using metadynamics, we mimic the effect of a single myosin head binding by determining the work required to pull small segments of tropomyosin toward the open position in several distinct regions of the thin filament. We find differences in required work due to the influence of cardiac troponin T lead to preferential binding sites and determine the mechanism of further myosin head recruitment.

TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding

Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

Single Molecule Visualization of Cardiac Myosin-Binding Protein C N-Terminal Fragments Interacting with Regulated Actin Filaments: Mechanisms of Calcium Sensitization

Tropomyosin movement on actin resulting from calcium binding to troponin permits myosin binding, filament sliding and force generation in the cardiac muscle sarcomere. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism is that cMyBP-C binds directly to the regulated actin filament at low calcium levels to move tropomyosin, thus recruiting myosin binding. To test this, we directly visualized fluorescently-labelled cMyBP-C N-terminal fragments and GFP-labelled myosin molecules binding to regulated actin filaments in a single molecule assay. Binding of cMyBP-C's C0C3 N-terminal domains to regulated actin enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 fragments bound cooperatively, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that utilizes a weak-binding mode to search for access to actin and a tight-binding mode that enhances myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to regulated actin filaments similarly to the Cy3-C0C3 fragment, the shorter fragments were unable to affect myosin binding. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

Spontaneous transitions of actin-bound tropomyosin toward blocked and closed states.

After muscle contraction, myosin cross-bridge heads detach from thin actin filaments during relaxation. Structural and kinetic data of cross-bridge-thin filament interactions have shown that tropomyosin's position on F-actin is biased toward the blocked or closed states when myosin detaches. It is not clear if structural linkages between tropomyosin and myosin cross-bridge heads, or tropomyosin and Ca2+-free troponin, drive the process or whether tropomyosin movement is energetically independent of myosin and troponin influence. Here we provide in silico data about tropomyosin dynamics on troponin/myosin-free F-actin indicating that tropomyosin moves from the open state toward blocked- or closed-state positions on actin. To follow transitions inherent to tropomyosin itself on F-actin, we performed MD simulations initiated from the blocked-, open-, and intermediate-state models and followed tropomyosin over the surface of F-actin in the absence of myosin and troponin. These MD simulations maintain tropomyosin in a cable-like conformation, including the tropomyosin overlap domain, while allowing tropomyosin to retain most of its motional freedom. Tropomyosin shows considerable azimuthal movement away from the open state toward the surrounds of a more energetically favorable blocked B-state position over F-actin. In contrast, little movement away from the B-state location is observed. Our results are consistent with previous predictions based on electrostatic interaction energy landscapes determined by rigid-body translocation of tropomyosin. They support the view that in the absence of myosin, i.e., when myosin cross-bridges detach from actin, the blocked- or closed-state positions of tropomyosin are energetically favored, while the open state is not.

Tropomyosin dynamics during cardiac muscle contraction as governed by a multi-well energy landscape

The dynamic oscillations of tropomyosin molecules in the azimuthal direction over the surface of the actin filament during thin filament activation are studied here from an energy landscape perspective. A mathematical model based on principles from nonlinear dynamics and chaos theory is derived to describe these dynamical motions. In particular, an energy potential with three wells is proposed to govern the tropomyosin oscillations between the observed regulatory positions observed during muscle contraction, namely the blocked “B”, closed “C” and open “M” states. Based on the variations in both the frequency and amplitude of the environmental (surrounding the thin filament system) driving tractions, such as the electrostatic, hydrophobic, and Ca2+-dependent forces, the tropomyosin movements are shown to be complex; they can change from being simple harmonic oscillations to being fully chaotic. Three cases (periodic, period-2, and chaotic patterns) are presented to showcase the different possible dynamic responses of tropomyosin sliding over the actin filament. A probability density function is used as a statistical measure to calculate the average residence time spanned out by the tropomyosin molecule when visiting each (B, C, M) equilibrium state. The results were found to depend strongly on the energy landscape profile and its featured barriers, which normally govern the transitions between the B-C-M states during striated muscle activation.

Effects of Myosin Inhibitors on the X-Ray Diffraction Patterns of Relaxed and Calcium-Activated Rabbit Skeletal Muscle Fibers

Two potent myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS) and blebbistatin, suppress the contractile force of skeletal muscle fibers at micromolar concentrations. To gain insights into the mechanism of inhibition by these chemicals, we studied their effects on the X-ray diffraction patterns from rabbit skeletal muscle fibers under relaxing and activating conditions. At 100 µM, both inhibitors almost completely suppress the contractile force, but still substantial changes were observed in the diffraction patterns upon calcium activation. (1) The 2nd actin layer line reflection was enhanced normally, indicating that calcium binding to troponin and the subsequent movement of tropomyosin are not inhibited, (2) the myosin layer line reflections became much weaker, and (3) the 1,1/1,0 intensity ratio of the equatorial reflections was increased. The observations (2) and (3) indicate that, even in the presence of the inhibitors at a high concentration, myosin heads leave the helix on the myosin filaments and approach the actin filaments. Interestingly, the d1,0 spacing of the filament lattice remains unchanged upon activation of inhibited fibers, in contrast to the case of normal activation in which the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, the pull is suppressed, and as a result, the heads simply bind to actin without exerting any force. The results support the idea that the inhibitors do not block the myosin binding to actin, but block the step of force-producing transition of the bound actomyosin complex.

CA2+-Induced Movement of Tropomyosin on Native Cardiac Thin Filaments Revealed by Cryoelectron Microscopy

CA2+-Induced Movement of Tropomyosin on Native Cardiac Thin Filaments Revealed by Cryoelectron Microscopy

Effects of myosin inhibitors on the X-ray diffraction patterns of relaxed and calcium-activated rabbit skeletal muscle fibers.

We studied the effect of myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS), blebbistatin, and butanedione monoxime (BDM) on X-ray diffraction patterns from rabbit psoas fibers under relaxing and contracting conditions. The first two inhibitors suppressed the contractile force almost completely at a 100 μM concentration, and a similar effect was obtained at 50 mM for BDM. However, still substantial changes were observed in the diffraction patterns upon calcium-activation of inhibited muscle fibers. (1) The 2nd actin layer-line reflection was enhanced normally, indicating that calcium binding to troponin and the subsequent movement of tropomyosin are not inhibited, (2) the myosin layer-line reflections became much weaker, and (3) the 1,1/1,0 intensity ratio of the equatorial reflections was increased. The observations (2) and (3) indicate that, even in the presence of the inhibitors at a saturating concentration, myosin heads leave the helix on the thick filaments and approach the thin filaments. Interestingly, the d1,0 spacing of the filament lattice remained unchanged upon activation of inhibited fibers, in contrast to the case of normal activation in which the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, the pull is suppressed, and as a result, the heads simply bind to actin without exerting any force. The results support the idea that the inhibitors do not block the myosin binding to actin, but block the step of force-producing transition of the bound actomyosin complex.

Molecular mechanisms of dysfunction of muscle fibres associated with Glu139 deletion in TPM2 gene

Deletion of Glu139 in β-tropomyosin caused by a point mutation in TPM2 gene is associated with cap myopathy characterized by high myofilament Ca2+-sensitivity and muscle weakness. To reveal the mechanism of these disorders at molecular level, mobility and spatial rearrangements of actin, tropomyosin and the myosin heads at different stages of actomyosin cycle in reconstituted single ghost fibres were investigated by polarized fluorescence microscopy. The mutation did not alter tropomyosin’s affinity for actin but increased strongly the flexibility of tropomyosin and kept its strands near the inner domain of actin. The ability of troponin to switch actin monomers “on” and “off” at high and low Ca2+, respectively, was increased, and the movement of tropomyosin towards the blocked position at low Ca2+ was inhibited, presumably causing higher Ca2+-sensitivity. The mutation decreased also the amount of the myosin heads which bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation; this may contribute to contractures and muscle weakness.

Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function

Elongated tropomyosin, associated with actin-subunits along the surface of thin filaments, makes electrostatic interactions with clusters of conserved residues, K326, K328, and R147, on actin. The association is weak, permitting low-energy cost regulatory movement of tropomyosin across the filament during muscle activation. Interestingly, acidic D292 on actin, also evolutionarily conserved, lies adjacent to the three-residue cluster of basic amino acids and thus may moderate the combined local positive charge, diminishing tropomyosin-actin interaction and facilitating regulatory-switching. Indeed, charge neutralization of D292 is connected to muscle hypotonia in individuals with D292V actin mutations and linked to congenital fiber-type disproportion. Here, the D292V mutation may predispose tropomyosin-actin positioning to a myosin-blocking state, aberrantly favoring muscle relaxation, thus mimicking the low-Ca2+ effect of troponin even in activated muscles. To test this hypothesis, interaction energetics and in vitro function of wild-type and D292V filaments were measured. Energy landscapes based on F-actin-tropomyosin models show the mutation localizes tropomyosin in a blocked-state position on actin defined by a deeper energy minimum, consistent with augmented steric-interference of actin-myosin binding. In addition, whereas myosin-dependent motility of troponin/tropomyosin-free D292V F-actin is normal, motility is dramatically inhibited after addition of tropomyosin to the mutant actin. Thus, D292V-induced blocked-state stabilization appears to disrupt the delicately poised energy balance governing thin filament regulation. Our results validate the premise that stereospecific but necessarily weak binding of tropomyosin to F-actin is required for effective thin filament function.

The reason for a high Ca2+-sensitivity associated with Arg91Gly substitution in TPM2 gene is the abnormal behavior and high flexibility of tropomyosin during the ATPase cycle

Substitution of Arg for Gly residue in 91th position in β-tropomyosin caused by a point mutation in TPM2 gene is associated with distal arthrogryposis, characterized by a high Ca2+-sensitivity of myofilament and contracture syndrome. To understand the mechanisms of this defect, we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres, using the polarized fluorescence microscopy. The mutation was shown to markedly decrease the bending stiffness of β-tropomyosin in the thin filaments. In the absence of the myosin heads the mutation did not alter the ability of troponin to shift tropomyosin to the blocked position and to switch actin monomers off at low Ca2+. During the ATPase cycle the movement of the mutant tropomyosin is restrained, it is located near the open position, which allows strong binding of the myosin heads to actin even at low Ca2+. This may be the reason for both high Ca2+-sensitivity and contractures associated with the Arg91Gly mutation. The use of reagents that decrease the Ca2+sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.

Distortion of the Actin A-Triad Results in Contractile Disinhibition and Cardiomyopathy

SUMMARYStriated muscle contraction is regulated by the movement of tropomyosin over the thin filament surface, which blocks or exposes myosin binding sites on actin. Findings suggest that electrostatic contacts, particularly those between K326, K328, and R147 on actin and tropomyosin, establish an energetically favorable F-actin-tropomyosin configuration, with tropomyosin positioned in a location that impedes actomyosin associations and promotes relaxation. Here, we provide data that directly support a vital role for these actin residues, termed the A-triad, in tropomyosin positioning in intact functioning muscle. By examining the effects of an A295S α-cardiac actin hypertrophic cardiomyopathy-causing mutation, over a range of increasingly complex in silico, in vitro, and in vivo Drosophila muscle models, we propose that subtle A-triad-tropomyosin perturbation can destabilize thin filament regulation, which leads to hypercontractility and triggers disease. Our efforts increase understanding of basic thin filament biology and help unravel the mechanistic basis of a complex cardiac disorder.

Ca2+-induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy

Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca2+ The current model of muscle regulation holds that at relaxing (low-Ca2+) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca2+) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca2+ activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF. We show that two of these tropomyosin positions restrain actomyosin interactions, whereas in the third position, which is significantly enhanced at high Ca2+, tropomyosin does not block myosin binding sites on F-actin. Our data provide a structural framework for the enhanced activation of the cardiac TF over the skeletal TF by Ca2+ and lead to a mechanistic model for the regulation of the cardiac TF.

Tropomyosin movement is described by a quantitative high-resolution model of X-ray diffraction of contracting muscle.

Contraction of skeletal and cardiac muscle is controlled by Ca2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca2+, Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the three-state model (McKillop and Geeves Biophys J 65:693-701, 1993), upon Ca2+ binding to Tn, Tpm rotates about the filament axis to a 'closed state' where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an 'open' position where neighboring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low-angle X-ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of X-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much-improved fit to the experimental data obtained from rabbit muscle fibers in relaxed and rigor states than previous lower-resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 to 0.3nm-1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin.