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.     

X-ray interference studies of crossbridge action in muscle contraction: evidence from quick releases

We have used a high-resolution small angle X-ray scattering system, together with a high-performance CCD camera, on the BioCAT beamline at the APS synchrotron radiation facility at the Argonne National Laboratory, to study X-ray interference effects in the meridional reflections generated by the arrays of myosin crossbridges in contracting muscle. These give information about axial movements of the myosin heads during contraction with sub-nanometer resolution. Using whole intact muscle preparations (frog sartorius) we have been able to record the detailed behavior of M3 (the first order meridional reflection from the myosin crossbridges, at 14.56 nm) at each of a number of quick releases of increasing magnitude, on the same specimen, and at the same time make similar measurements on higher order myosin meridional reflections, particularly M6. The latter provides information about the dispersion of lever arm angles of the actin-attached myosin heads. The observations show that in isometric contraction the lever arm angles are dispersed through +/- 20-25 degrees on either side of a mean orientation that is about 60 degrees away from their orientation at the end of the working stroke: and that they move towards that orientation in synchronized fashion, with constant dispersion, during quick releases. The relationship between the shift in the interference fringes (which measures the shift of the myosin heads scattering mass towards the center of the sarcomere, and the changes in the total intensity of the reflections, which measures the changes in the axial profile of the heads, is consistent with the tilting lever arm mechanism of muscle contraction. Significant fixed contributions to the meridional reflections come from unattached myosin heads and from backbone components of the myosin filaments, and the interaction of these with the contributions from actin-attached myosin heads determines the behavior of these reflections.     

A mathematical model of the sarcomere mechanics of cross-striated muscle with consideration of the stretch and twist of actin filaments

A mathematical model of sarcomere mechanics, which takes into account the elongation of actin and myosin filaments and also twisting of the actin filaments in the sarcomere of striated muscle during contraction is presented. The model accounts for the experimentally observed phenomena of the stretch and twist of the actin filaments due to strong binding of myosin heads and the pulling force. Some model parameters were estimated from published experimental data. The results of modeling show that the twist of actin filaments can play a substantial role in the mechanical responses of contracting muscle fibers to step changes of their length.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

A mathematical model for sarcomere mechanics in cross-striated muscles taking into account the stretch and twist of actin filaments

A model of sarcomere mechanics, which takes into account the elongation of the actin and myosin filaments and twisting of the actin filaments during muscle contraction is suggested. The model accounts for the experimentally observed phenomena of the stretch and twist of actin filaments due to strong binding of myosin heads and pulling force. Some model parameters were estimated from published experimental data. The results of modelling suggest that the twist of actin filaments may play an essential role in mechanical responses of contracting muscle fibres to stepwise changes in their length.     

The Fraction of Myosin Motors That Participate in Isometric Contraction of Rabbit Muscle Fibers at Near-Physiological Temperature

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca²⁺ at 4°C were heated to 31–34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41–43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ∼6.3 pN.     

The conformation of myosin head domains in rigor muscle determined by X-ray interference

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T(0)), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T(0)), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T(0), the heads' center of mass moved back by 1.1-1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys(707)-Lys(843) vector and the filament axis increases by approximately 36 degrees between isometric contraction and low-force rigor, and decreases by 7-10 degrees when the rigor fiber is stretched to 0.55 T(0).     

Myosin filament sliding through the Z-disc relates striated muscle fibre structure to function

Striated muscle contraction requires intricate interactions of microstructures. The classic textbook assumption that myosin filaments are compressed at the meshed Z-disc during striated muscle fibre contraction conflicts with experimental evidence. For example, myosin filaments are too stiff to be compressed sufficiently by the muscular force, and, unlike compressed springs, the muscle fibres do not restore their resting length after contractions to short lengths. Further, the dependence of a fibre''s maximum contraction velocity on sarcomere length is unexplained to date. In this paper, we present a structurally consistent model of sarcomere contraction that reconciles these findings with the well-accepted sliding filament and crossbridge theories. The few required model parameters are taken from the literature or obtained from reasoning based on structural arguments. In our model, the transition from hexagonal to tetragonal actin filament arrangement near the Z-disc together with a thoughtful titin arrangement enables myosin filament sliding through the Z-disc. This sliding leads to swivelled crossbridges in the adjacent half-sarcomere that dampen contraction. With no fitting of parameters required, the model predicts straightforwardly the fibre''s entire force–length behaviour and the dependence of the maximum contraction velocity on sarcomere length. Our model enables a structurally and functionally consistent view of the contractile machinery of the striated fibre with possible implications for muscle diseases and evolution.     

Dynamics of myosin-driven skeletal muscle contraction: I. Steady-state force generation

Skeletal muscle contraction is a canonical example of motor-driven force generation. Despite the long history of research in this topic, a mechanistic explanation of the collective myosin force generation is lacking. We present a theoretical model of muscle contraction based on the conformational movements of individual myosins and experimentally measured chemical rate constants. Detailed mechanics of the myosin motor and the geometry of the sarcomere are taken into account. Two possible scenarios of force generation are examined. We find only one of the scenarios can give rise to a plausible contraction mechanism. We propose that the synchrony in muscle contraction is due to a force-dependent ADP release step. Computational results of a half sarcomere with 150 myosin heads can explain the experimentally measured force-velocity relationship and efficiency data. We predict that the number of working myosin motors increases as the load force is increased, thus showing synchrony among myosin motors during muscle contraction. We also find that titin molecules anchoring the thick filament are passive force generators in assisting muscle contraction.     

Changes in the X-ray reflections from contracting muscle during rapid mechanical transients and their structural implications

During normal contractions of vertebrate striated muscle, it is believed that the cross-bridges which produce the sliding force undergo asynchronous cyclical changes in their structure. Thus, an X-ray diffraction diagram from a muscle under these conditions will give structural information averaged over the whole range of cross-bridge states. Such diagrams show characteristic and informative differences from those given by relaxed muscle, but can give little information about changes in the configuration of the cross-bridges at different stages of their working stroke. However, it is possible to effect a partial synchronization of these changes by applying very rapid changes in length, completed in less than one millisecond to an otherwise isometrically contracting muscle. If the amplitude of these length changes is comparable to the length of the cross-bridge stroke (say 100 A per half-sarcomere), then it should bring about a transient but significant redistribution of cross-bridge states, which would show up in the X-ray diagram. We have made use of synchrotron radiation as a high intensity X-ray source in order to record such patterns with the necessary time resolution (1 ms or less) and have found major changes in the intensity of the 143 A meridional reflection accompanying the rapid length changes of the muscle. These changes appear to arise from specific configurational changes in the cross-bridges during the working stroke. A model is suggested in which the 143 A meridional intensity in a contracting muscle arises mainly from attached cross-bridges and is generated by the part of the myosin head near the S1-S2 junction. During normal contraction, cross-bridges go through their structural cycle asynchronously with each other, since they start at different times, but if the S2 changes in length rather little, then the configurational changes in the myosin heads are synchronized with the actin filament movement in such a way that the S1-S2 junction remains relatively fixed in its axial position. In a quick release, it is suggested that bringing many S1 heads simultaneously to the end of their working strokes on actin disrupts the 143 A axial repeat of their distal ends near S2, and brings about the large decrease of the 143 A meridional reflection. This model therefore involves a large change in the position of part of the myosin head structure relative to actin during the working stroke of the cross-bridge.     

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.     

Behaviour of the crossbridges in stretched or compressed muscle fibres

The maximum chord of the myosin heads is comparable to the closest surface-to-surface spacing between the myofilaments in a muscle at the slack length. Therefore, when the sarcomere length increases or when the fibre is compressed, the surface-to-surface myofilament spacing becomes lower than the head long axis. We conclude that, in stretched or compressed fibres, some crossbridges cannot attach, owing to steric hindrance. When the amount of compression is limited, this hindrance may be overcome by a tilting of the heads in the plane perpendicular to the filament axes; in this case, there is no consequence as concerns the crossbridge properties. In highly compressed fibres, the crossbridges become progressively hindered and all the crossbridges are hindered for an axis-to-axis spacing representing about 60% of the spacing observed under zero external osmotic pressure. In this case, both the isometric tension and the ATPase activity of the fibre are zero. In fibres stretched up to 3.77 microns (sarcomere length corresponding to the disappearance of the overlap between the thick and the thin filaments), the ratio of hindered crossbridges over the functional crossbridges may be estimated at about 55%. In stretched fibres, a noticeable proportion of crossbridges are sterically hindered and the crossbridges performance (e.g. constants of attachment and detachment) depends on filament spacing, i.e. on sarcomere length. Therefore, we think it is probably impossible to consider the crossbridges as independent force converters, since this idea requires that the crossbridge properties are independent of sarcomere length. In this connection, all the experiments performed on osmotically compressed fibres are of major importance for the understanding of the true mechanisms of muscle contraction.     

Cross-bridge number, position, and angle in target zones of cryofixed isometrically active insect flight muscle

Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.5-nm-spaced shelves along the thick filament to their thin filament attachments in the target zones. The relationship between the probability of cross-bridge formation and axial offset between the shelf and target zone center was well fitted by a Gaussian distribution. One head of each myosin whose origin is close to an actin target zone forms a cross-bridge most of the time. The probability of cross-bridge formation remains high for myosin heads originating within 8 nm axially of the target zone center and is low outside 12 nm. We infer that most target zone cross-bridges are nearly perpendicular to the filaments (60% within 11 degrees ). The results suggest that in isometric contraction, most cross-bridges maintain tension near the beginning of their working stroke at angles near perpendicular to the filament axis. Moreover, in the absence of filament sliding, cross-bridges cannot change tilt angle while attached nor reach other target zones while detached, so may cycle repeatedly on and off the same actin target monomer.     

3D structure of relaxed fish muscle myosin filaments by single particle analysis

To understand the structural changes involved in the force-producing myosin cross-bridge cycle in vertebrate muscle it is necessary to know the arrangement and conformation of the myosin heads at the start of the cycle (i.e. the relaxed state). Myosin filaments isolated from goldfish muscle under relaxing conditions and viewed in negative stain by electron microscopy (EM) were divided into segments and subjected to three-dimensional (3D) single particle analysis without imposing helical symmetry. This allowed the known systematic departure from helicity characteristic of vertebrate striated muscle myosin filaments to be preserved and visualised. The resulting 3D reconstruction reveals details to about 55 A resolution of the myosin head density distribution in the three non-equivalent head 'crowns' in the 429 A myosin filament repeat. The analysis maintained the well-documented axial perturbations of the myosin head crowns and revealed substantial azimuthal perturbations between crowns with relatively little radial perturbation. Azimuthal rotations between crowns were approximately 60 degrees , 60 degrees and 0 degrees , rather than the regular 40 degrees characteristic of an unperturbed helix. The new density map correlates quite well with the head conformations analysed in other EM studies and in the relaxed fish muscle myosin filament structure modelled from X-ray fibre diffraction data. The reconstruction provides information on the polarity of the myosin head array in the A-band, important in understanding the geometry of the myosin head interaction with actin during the cross-bridge cycle, and supports a number of conclusions previously inferred by other methods. The observed azimuthal head perturbations are consistent with the X-ray modelling results from intact muscle, indicating that the observed perturbations are an intrinsic property of the myosin filaments and are not induced by the proximity of actin filaments in the muscle A-band lattice. Comparison of the axial density profile derived in this study with the axial density profile of the X-ray model of the fish myosin filaments which was restricted to contributions from the myosin heads allows the identification of a non-myosin density peak associated with the azimuthally perturbed head crown which can be interpreted as a possible location for C-protein or X-protein (MyBP-C or -X). This position for C-protein is also consistent with the C-zone interference function deduced from previous analysis of the meridional X-ray pattern from frog muscle. It appears that, along with other functions, C-(X-) protein may have the role of slewing the heads of one crown so that they do not clash with the neighbouring actin filaments, but are readily available to interact with actin when the muscle is activated.     

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.     

Myosin filament polymerization and depolymerization in a model of partial length adaptation in airway smooth muscle

Length adaptation in airway smooth muscle (ASM) is attributed to reorganization of the cytoskeleton, and in particular the contractile elements. However, a constantly changing lung volume with tidal breathing (hence changing ASM length) is likely to restrict full adaptation of ASM for force generation. There is likely to be continuous length adaptation of ASM between states of incomplete or partial length adaption. We propose a new model that assimilates findings on myosin filament polymerization/depolymerization, partial length adaptation, isometric force, and shortening velocity to describe this continuous length adaptation process. In this model, the ASM adapts to an optimal force-generating capacity in a repeating cycle of events. Initially the myosin filament, shortened by prior length changes, associates with two longer actin filaments. The actin filaments are located adjacent to the myosin filaments, such that all myosin heads overlap with actin to permit maximal cross-bridge cycling. Since in this model the actin filaments are usually longer than myosin filaments, the excess length of the actin filament is located randomly with respect to the myosin filament. Once activated, the myosin filament elongates by polymerization along the actin filaments, with the growth limited by the overlap of the actin filaments. During relaxation, the myosin filaments dissociate from the actin filaments, and then the cycle repeats. This process causes a gradual adaptation of force and instantaneous adaptation of shortening velocity. Good agreement is found between model simulations and the experimental data depicting the relationship between force development, myosin filament density, or shortening velocity and length.     

Geometrical factors influencing muscle force development. I. The effect of filament spacing upon axial forces.

The influence of geometry on the force and stiffness measured during muscle contraction at different sarcomere lengths is examined by using three specific models of muscle cross-bridge geometry which are based upon the double-hinge model of H. E. Huxley (Science [Wash. D.C.]. 1969, 164:1356-1366) extended to three dimensions. The force generated during muscle contraction depends upon the orientation of the individual cross-bridge force vectors and the distribution of the cross-bridges between various states. For the simplest models, in which filament separation has no effect upon cross-bridge distribution, it is shown that changes in force vectors accompanying changes in myofilament separation between sarcomere lengths 2.0 and 3.65 microgram in an intact frog skeletal muscle fiber have only a small effect upon axial force. The simplest models, therefore, produce a total axial force proportional to the overlap between the actin and myosin filaments and independent of filament separation. However, the analysis shows that it is possible to find assumptions that produce a cross-bridge model in which the axial force is not independent of filament spacing. It is also shown that for some modes of attachment of subfragment-1 (S1) to actin the azimuthal location of the actin site is important in determining the axial force. A mode of S1 attachment to actin similar to that deduced by Moore et al. (J. Mol. Biol., 1970, 50:279-294), however, exhibits rather constant cross-bridge behavior over a wide range of actin site location.     

Motor Force Homeostasis in Skeletal Muscle Contraction

In active biological contractile processes such as skeletal muscle contraction, cellular mitosis, and neuronal growth, an interesting common observation is that multiple motors can perform coordinated and synchronous actions, whereas individual myosin motors appear to randomly attach to and detach from actin filaments. Recent experiment has demonstrated that, during skeletal muscle shortening at a wide range of velocities, individual myosin motors maintain a force of ∼6 pN during a working stroke. To understand how such force-homeostasis can be so precisely regulated in an apparently chaotic system, here we develop a molecular model within a coupled stochastic-elastic theoretical framework. The model reveals that the unique force-stretch relation of myosin motor and the stochastic behavior of actin-myosin binding cause the average number of working motors to increase in linear proportion to the filament load, so that the force on each working motor is regulated at ∼6 pN, in excellent agreement with experiment. This study suggests that it might be a general principle to use catch bonds together with a force-stretch relation similar to that of myosin motors to regulate force homeostasis in many biological processes.     

Structural features of cross-bridges in isometrically contracting skeletal muscle

Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1 nm and of the first actin layer line at 37 nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATPgammaS at saturating [Ca2+] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge.