Changes of thick filament structure during contraction of frog striated muscle.

The strongest myosin-related features in the low-angle axial x-ray diffraction pattern of resting frog sartorius muscle are the meridional reflections corresponding to axial spacings of 21.4 and 14.3 nm, and the first layer line, at a spacing 42.9 nm. During tetanus the intensities of the first layer line and the 21.4-nm meridional decrease by 62 and 80% respectively, but, when the muscle is fresh, the 14.3-nm meridional intensity rises by 13%, although it shows a decrease when the muscle is fatigued. The large change in the intensity of the 21.4-nm meridional reflection suggests that the projected myosin cross-bridge density onto the thick filament axis changes during contraction. The model proposed by Bennett (Ph.D. Thesis, University of London, 1977) in which successive cross-bridge levels are at 0,3/8, and 5/8 of the 42.9-nm axial repeat in the resting muscle, passing to 0, 1/3, and 2/3 in the contracting state, can explain why the 21.4-nm reflection decreases in intensity while the 14.3-nm increases when the muscle is activated. The model predicts a rather larger increase of the 14.3-nm reflection intensity during contraction than that observed, but the discrepancy may be removed if a small change of shape or tilt of the cross-bridges relative to the thick filament axis is introduced. The decrease of the intensity of the first layer line indicates that the cross-bridges become disordered in the plane perpendicular to the filament axis.     

An X-Ray Diffraction Study on Early Structural Changes in Skeletal Muscle Contraction

Structural changes in frog skeletal muscle were studied using x-ray diffraction with a time resolution of 0.53–1.02 ms after a single electrical stimulus at 8°C. Tension began to drop at 6 ms (latency relaxation), reached a minimum at 8 ms, and then twitch tension developed. The intensity of the meridional reflection at 1/38.5 nm⁻¹, from troponin molecules on the thin filament, began to increase at 4–5 ms and reached a maximum at ~12 ms. The meridional reflections based on the myosin 43-nm repeat began to decrease when the tension began to develop. The peak position of the third-order myosin meridional reflection began to shift toward the higher angle at ~5 ms, reached a maximum shift (0.02%) at 10 ms, and then moved toward the lower angle. The intensity of the second actin layer line at 1/18 nm⁻¹ in the axial direction, which was measured at 12°C, began to rise at 5 ms, whereas the latency relaxation started at 3.5 ms. These results suggest that 1), the Ca²⁺-induced structural changes in the thin filament and a structural change in the thick filament have already taken place during latency relaxation; and 2), the Ca²⁺ regulation of the thin filament is highly cooperative.     

Time-resolved x-ray study of effect of sinusoidal length change on tetanized frog muscle.

Time-resolved x-ray diffraction studies were done on frog skeletal muscles with synchrotron radiation by applying sinusoidal length changes of frequency 10 Hz and amplitude approximately 1% to isometrically contracting muscles at approximately 17 degrees C. Distinct periodic intensity changes were observed in the 14.3-nm myosin meridional reflection and the equatorial 1,0 and 1,1 reflections. Response of the 14.3-nm reflection to the sinusoidal length change was nonlinear, as evidenced by a large second harmonic in its oscillatory intensity change, whereas the response of the equatorial 1,1 reflection was closely linear, as evidenced by almost sinusoidal intensity change. Intensity change of the 1,0 reflection was nearly antiphase to that of the 1,1 reflection. Integral widths of the 14.3-nm meridional reflection measured along the meridian and of the equatorial 1,1 reflection remained almost constant during tension development, while that of the 1,0 reflection tended to decrease. The widths of the 14.3-nm meridional reflection perpendicular to the meridian and of the equatorial 1,0 reflection appeared to undergo oscillatory changes in response to the sinusoidal length changes.     

Structural changes of cross-bridges on transition from isometric to shortening state in frog skeletal muscle

Structural changes in the myosin cross-bridges were studied by small-angle x-ray diffraction at a time resolution of 0.53 ms. A frog sartorius muscle, which was electrically stimulated to induce isometric contraction, was released by approximately 1% in 1 ms, and then its length was decreased to allow steady shortening with tension of approximately 30% of the isometric level. Intensity of all reflections reached a constant level in 5-8 ms. Intensity of the 7.2-nm meridional reflection and the (1,0) sampling spot of the 14.5-nm layer line increased after the initial release but returned to the isometric level during steady shortening. The 21.5-nm meridional reflection showed fast and slow components of intensity increase. The intensity of the 10.3-nm layer line, which arises from myosin heads attached to actin, decreased to a steady level in 2 ms, whereas other reflections took longer, 5-20 ms. The results show that myosin heads adapt quickly to an altered level of tension, and that there is a distinct structural state just after a quick release.     

Structural changes of actin-bound myosin heads after a quick length change in frog skeletal muscle

Changes in the x-ray diffraction pattern from a frog skeletal muscle were recorded after a quick release or stretch, which was completed within one millisecond, at a time resolution of 0.53 ms using the high-flux beamline at the SPring-8 third-generation synchrotron radiation facility. Reversibility of the effects of the length changes was checked by quickly restoring the muscle length. Intensities of seven reflections were measured. A large, instantaneous intensity drop of a layer line at an axial spacing of 1/10.3 nm(-1) after a quick release and stretch, and its partial recovery by reversal of the length change, indicate a conformational change of myosin heads that are attached to actin. Intensity changes on the 14.5-nm myosin layer line suggest that the attached heads alter their radial mass distribution upon filament sliding. Intensity changes of the myosin reflections at 1/21.5 and 1/7.2 nm(-1) are not readily explained by a simple axial swing of cross-bridges. Intensity changes of the actin-based layer lines at 1/36 and 1/5.9 nm(-1) are not explained by it either, suggesting a structural change in actin molecules.     

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.     

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.     

Structural changes in the muscle thin filament during contractions caused by single and double electrical pulses

In order to investigate the structural changes of the myofilaments involved in the phenomenon of summation in skeletal muscle contraction, we studied small-angle x-ray intensity changes during twitches of frog skeletal muscle elicited by either a single or a double stimulus at 16 °C. The separation of the pulses in the double-pulse stimulation was either 15 or 30 ms. The peak tension was more than doubled by the second stimulus. The equatorial (1,0) intensity, which decreased upon the first stimulus, further decreased with the second stimulus, indicating that more cross-bridges are formed. The meridional reflections from troponin at 1/38.5 and 1/19.2 nm^− 1 were affected only slightly by the second stimulus, showing that attachment of a small number of myosin heads to actin can make a cooperative structural change. In overstretched muscle, the intensity increase of the troponin reflection in response to the second stimulus was smaller than that to the first stimulus. These results show that the summation is not due to an increased Ca binding to troponin and further suggest a highly cooperative nature of the structural changes in the thin filament that are related to the regulation of contraction.     

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by ∼0.1% upon activation relative to the relaxing state and increased by ∼0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca²⁺-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca²⁺-binding and the second induced by actomyosin interaction.     

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.     

Time-resolved X-ray diffraction studies of frog skeletal muscle isometrically twitched by two successive stimuli using synchrotron radiation

In order to clarify the delay between muscular structural changes and mechanical responses, the intensity changes of the equatorial and myosin layer-line reflections were studied by a time-resolved X-ray diffraction technique using synchrotron radiation. The muscle was stimulated at 12-13 degrees C by two successive stimuli at an interval (80-100 ms) during which the second twitch started while tension was still being exerted by the muscle. At the first twitch, the intensity changes of the 1.0 and 1.1 equatorial reflections reached 65 and 200% of the resting values, and further changes to 55 and 220% were seen at the second twitch, respectively. Although the second twitch decreased not only the time to peak tension but also that to the maximum intensity changes of the equatorial reflections (in both cases, about 15 ms), the delay (about 20 ms) between the intensity changes and the development of tension at the first twitch were still observed at the second twitch. On the other hand, the intensities of the 42.9 nm off-meridional and the 21.5 nm meridional myosin reflections decreased at the first twitch to the levels found when a muscle was isometrically tetanized, and no further decrease in their intensities was observed at the second twitch. These results indicate that a certain period of time is necessary for myosin heads to contribute to tension development after their arrival in the vicinity of the thin filaments during contraction.     

Time-resolved x-ray diffraction studies on the intensity changes of the 5.9 and 5.1 nm actin layer lines from frog skeletal muscle during an isometric tetanus using synchrotron radiation.

Time-resolved x-ray diffraction studies have been made on the 5.9- and 5.1-nm actin layer lines from frog skeletal muscles during an isometric tetanus at 6 degrees C, using synchrotron radiation. The integrated intensities of these actin layer lines were found to increase during a tetanus by 30-50% for the 5.9-nm reflection and approximately 70% for the 5.1-nm reflection of the resting values. The intensity increase of both reflections was greater than that taking place in the transition from rest to rigor state. The intensity change of the 5.9-nm reflection preceded those of the myosin 42.9-nm off-meridional reflection and of the equatorial reflections, as well as the isometric tension development. The intensity profile of the 5.9-nm layer line during contraction was found to be different from that observed in the rigor state.     

Monitoring the Structural Behavior of Troponin and Myoplasmic Free Ca Concentration during Twitch of Frog Skeletal Muscle

The interaction of troponin molecules on the thin filament with Ca²⁺ plays a key role in regulating muscle contraction. To characterize the structural changes of troponin caused by Ca²⁺ and crossbridge formation, we recorded the small-angle x-ray intensity and the myoplasmic free Ca²⁺ concentration using fluo-3 AM in the same frog skeletal muscle during twitch elicited by a single electrical pulse at 16°C. In an overstretched muscle, the intensity of the meridional reflection from troponin at 1/38.5 nm⁻¹ began to change at 4 ms after the stimulus, reached a peak at 10 ms, and returned to the resting level with a halftime of 25 ms. The concentration of troponin-bound Ca²⁺ began to increase at 1-2 ms after the stimulus, reached a peak at 5 ms, and returned to the resting level with a halftime of 40 ms, indicating that troponin begins to change conformation only after a sizable amount of Ca²⁺ has bound to it, and returns to the resting structure even when there is still some bound Ca²⁺. In a muscle with a filament overlap, crossbridge formation appears to slow down Ca²⁺ release from troponin and have a large effect on its conformation.     

Changes in the X-ray diffraction pattern from rigor muscles by application of external length changes

The effect of external force on the X-ray pattern from frog muscles in rigor was studied by a time-resolved diffraction technique. When sinusoidal length changes (1.5–3% of the muscle length, 5Hz) were applied to the muscle, the 14.3 nm intensity decreased during the releasing phase and increased during the stretching phase. The intensity ratio of the equatorial 1,0 and 1,1 reflections did not change, nor were there any appreciable intensity changes in the 5.9 nm and 5.1 nm reflections during the length change. Experiments were also done with the relaxed muscles and no change was seen in any reflection, indicating that the rigor linkages are needed to produce the 14.3 nm intensity change. Thus the distinct effect of the length change was detected only on the 14.3 nm reflection. These results suggest no large conformational changes are induced in both the distal part of the myosin head attached to actin and the actin filament during the oscillation. It is therefore most probable that the proximal portion of myosin heads including S-2 contributes to the intensity change in response to the length change (see, also ref.21). When the muscle was stretched beyond the filament overlap, the 14.3 nm intensity change was suppressed to less than 50% of that of the slack length. It was also found that the tension change delayed the intensity change during the length oscillation. However, this delay of the tension change as observed in the muscle at the slack length was lacking in the overstretched muscle, indicating that the 14.3 nm intensity change may arise partly from a portion other than the crossbridges.     

The mammalian cardiac muscle thick filament: backbone contributions to meridional reflections

Information about the structure of the vertebrate striated muscle thick filament backbone is important for understanding the arrangement of both the rod portion of the myosin molecule and the accessory proteins associated with the backbone region of the filament. Although models of the backbone have been proposed, direct data on the structure of the backbone is limited. In this study, we provide evidence that electron micrographs of isolated negatively stained cardiac thick filaments contain significant information about the filament backbone. Computed Fourier transforms from isolated cardiac thick filaments show meridional (or near meridional) reflections on the 10th and 11th layer lines that are particularly strong. Comparison of Fourier filtrations of the filaments that exclude, or include, these reflections, provide evidence that these reflections originate at least in part from a series of striations on the backbone at a approximately 4 nm spacing. The striations are likely to result either from the packing of the myosin rods, or from proteins such as titin associated with the filament backbone.     

Understanding the organisation and role of myosin binding protein C in normal striated muscle by comparison with MyBP-C knockout cardiac muscle

Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58 ± 0.01 μm. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra “forbidden” meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.     

Structural changes during activation of frog muscle studied by time-resolved X-ray diffraction

The pattern given by contracting frog muscle can be followed with high time resolution using synchrotron radiation as a high-intensity X-ray source. We have studied the behaviour of the second actin layer-line (axial spacing of approximately 179 A) at an off-meridional spacing of approximately 0.023 A-1, a region of the diagram that is sensitive to the position of tropomyosin in the thin filaments. In confirmation of earlier work, we find that there is a substantial increase in the intensity of this part of the pattern during contraction. We find that the reflection reaches half its final intensity about 17 milliseconds after the stimulus at 6 degrees C. The changes in the equatorial reflections, which arise from movement of crossbridges towards the thin filaments, occur with a delay of about 12 to 17 milliseconds relative to this change in the actin pattern. In over-stretched muscle, where thick and thin filaments no longer overlap, the changes in the actin second layer-line still take place upon stimulation with a time course and intensity similar to that observed at full overlap. This indicates that tropomyosin movement, in response to calcium binding to troponin, is the first structural step in muscular contraction, and is the prerequisite for myosin binding. A change in intensity similar to that found in contracting muscle is seen in rigor, where tropomyosin is probably locked in the active position. During relaxation the earlier stages in the decrease in intensity of the second actin layer-line take place significantly sooner after the last stimulus than tension decay. In over-stretched muscles the intensity decay is appreciably faster than in the same muscles at rest length, where attached crossbridges may interfere with the return of tropomyosin to its resting position.     

Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a “nanosurfer” assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human β-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0–C2 or C1–C2) on nanotubes every 14 nm. Both C0–C2 and C1–C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0–C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0–C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of β-cardiac myosin contractility.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.