Lateral forces in the filament lattice of vertebrate striated muscle in the rigor state.

The repulsive pressure between filaments in the lattice of skinned rabbit and frog striated muscle in rigor has been measured as a function of interfilament spacing, using the osmotic pressure generated by solutions of large, uncharged polymeric molecules (dextran and polyvinylpyrrolidone). The pressure/spacing measurements have been compared with theoretically derived curves for electrostatic pressure. In both muscles, the major part of the experimental curves (100-2,000 torr) lies in the same region as the electrostatic pressure curves, providing that a thick filament charge diameter of approximately 30 nm in rabbit and approximately 26 nm in frog is assumed. In chemically skinned or glycerol-extracted rabbit muscle the fit is good; in chemically skinned frog sartorius and semitendinosus muscle the fit is poor, particularly at lower pressures where a greater spacing is observed than expected on theoretical grounds. The charge diameter is much larger than the generally accepted value for thick filament backbone diameter. This may be because electron microscope results have underestimated the amount of filament shrinkage during sample preparation, or because most of the filament charge is located at some distance from the backbone surface, e.g., on HMM-S2. Decreasing the ionic strength of the external solution, changing the pH, and varying the sarcomere length all give pressure/spacing changes similar to those expected from electrostatic pressure calculations. We conclude that over most of the external pressure range studied, repulsive pressure in the lattice is predominantly electrostatic.     

Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

Effects of hyperosmotic solutions on the filament lattice of intact frog skeletal muscle.

The effect of increasing the osmotic strength of the extracellular solution on the fifament lattice of living frog sartorius and semitendinosus muscle has been studied using low-angle x-ray diffraction to measure the lattice spacing. As the extracellular osmotic strength is increased, the filament lattice shrinks like an osmometer until a minimal spacing between the thick filaments is reached. This minimal spacing varies from 20 to 31 nm, depending on the sarcomere length. Further increase in the osmotic strength produces little further shrinkage. The osmotic shrinkage curve indicates, for both muscles, an osmotically-inactive volume of approximately 30% of the volume in normal Ringer's solution. Shrinkage appears to be independent of temperature and the type of particle used to increase the osmotic strength (glucose, sucrose, small ions). The rate at which osmotic equilibruim is reached depends on muscle size, being slower for greater muscle diameters. Equilibrium spacings are approached exponentially with time constants ranging from 20 to 60 min. Independent of osmotic equilibrium, the lattice tends to shrink slowly by approximately 3% over the first few hours after dissection, probably because of a leakage of K+ ions from inside the muscle cells. This can be partly prevented by using an extracellular solution which contains a higher concentration of K+ ions or which is hypoosmotic. The volume of the muscle filament lattice (1.155d10(2) . S) is constant over a very wide range of sarcomere lengths, and is equal to approximately 3.6 x 10(6) nm3 for a range of amphibian muscle types.     

Time-resolved equatorial X-ray diffraction studies of skinned muscle fibres during stretch and release.

Equatorial X-ray diffraction patterns were recorded from small bundles of one to three chemically skinned frog sartorius muscle fibres (time resolution 250 microseconds) during rapid stretch and subsequent release. In the relaxed state, the dynamic A-band lattice spacing change as a result of a 2 % step stretch (determined from the positions of the 10 and 11 reflections) resulted in a 21 % increase in lattice volume, while static studies of spacing and sarcomere length indicated than an increase in volume of >/=50 % for the same length change. In rigor, stretch caused a lattice volume decrease which was reversed by a subsequent release. In activated fibres (pCa 4.5) exposed to 10 mM 2,3-butanedione 2-monoxime (BDM), stretch was accompanied by a lattice compression exceeding that of constant volume behaviour, but during tension recovery, compression was partially reversed to leave a net spacing change close to that observed in the relaxed fibre. In the relaxed state, spacing changes were correlated with the amplitude of the length step, while in rigor and BDM states, spacing changes correlated more closely with axial force. This behaviour is explicable in terms of two components of radial force, one due to structural constraints as seen in the relaxed state, and an additional component arising from cross-bridge formation. The ratio of axial to radial force for a single thick filament resulting from a length step was four in rigor and BDM, but close to unity for the relaxed state.     

The myofilament lattice: studies on isolated fibers. IV. Lattice equilibria in striated muscle.

Accounts of similarities between the thick filament lattice of striated muscle and smectic liquid-crystalline structures have focused upon an equilibrium between electrostatic (repulsive) and van der Waal's (attractive) forces. In living, intact muscle the fiber volume constitutes an additional important parameter which influences the amount of interaxial separation between the filaments. This is demonstrable by comparison of the lattice behavior of living fibers with that of fibers from which the sarcolemma has either been removed or made leaky by glycerination. These comparisons were made mainly by low-angle X-ray diffraction under conditions of changes in sarcomere length, ionic strength or osmolarity, and pH. Single fibers with the sarcolemma removed and glycerinated muscle have lattices which behave in accord with equilibrium liquid-crystalline systems in which the thick filament spacing is determined by the balance between electrostatic and van der Waal's forces. Conversely, osmotic and shortening studies demonstrate that the living, intact muscle has a lattice which behaves in accord with the so-called non-equilibrium (volume-constrained) liquid-crystalline condition in which the interaxial separation between the thick filaments is solely due to the amount of volume available as determined by the Donnan steady-state across the sarcolemma.     

Changes in the lateral filament spacing of skinned muscle fibres when cross-bridges attach

When a skinned fibre prepared from frog skeletal muscle goes from the relaxed to the rigor state at a sarcomere length of about 2.2 μm, the 1, 0 transverse spacing of the filament lattice, measured by X-ray diffraction, decreases by about 11%. In measurements at various sarcomere lengths, the decrease in the spacing was approximately proportional to the degree of overlap between the thick and thin filaments. This suggests that the shrinkage of the lattice is caused by a lateral force produced by cross-bridges. In order to estimate the magnitude of the lateral force, the decrease of spacing between relaxed and rigor states was compared with the shrinkage caused osmotically by adding a high molecular weight polymer, polyvinylpyrrolidone, to the bathing solution. The results indicate that the lateral force produced per unit length of thick filament in the overlap zone is of the same order of magnitude as the axially directed force produced during maximum isometric contraction (10^?10 to 10^?9 N/μm).Experiments in the presence of a high concentration of polyvinylpyrrolidone (100 g/l) show that when the lattice spacing is decreased osmotically beyond a certain value, the lateral force produced when the fibre goes into rigor changes its direction, causing the lattice to swell. This result can be explained by assuming that there is an optimum interfilament spacing at which the cross-bridges produce no lateral force. At other spacings, the lateral force tends to displace the filament lattice toward that optimum value.     

Tension fluctuations in contracting myofibrils and their interpretation

Repulsive pressure has been measured as a function of lattice spacing in gels of tobacco mosaic virus (TMV) and in the filament lattice of vertebrate striated muscle. External pressures up to ten atm have been applied to these lattices by an osmotic stress method. Numerical solutions to the Poisson-Boltzmann equation in hexagonal lattices have been obtained and compared to the TMV and muscle data. The theoretical curves using values for k calculated from the ionic strength give a good fit to experimental data from TMV gels, and an approximate fit to that from the muscle lattice, provided that a charge radius for the muscle thick filaments of approximately 16 nm is assumed. Variations in ionic strength, sarcomere length and state of the muscle give results which agree qualitatively with the theory, though a good fit between experiment and theory in the muscle case will clearly require consideration of other types of forces. We conclude that Poisson-Boltzmann theory can provide a good first approximation to the long-range electrostatic forces operating in such biological gel systems.     

An electrostatic model with weak actin-myosin attachment resolves problems with the lattice stability of skeletal muscle

The stability of the filament lattice in relaxed striated muscle can be viewed as a balance of electrostatic and van der Waals forces. The simplest electrostatic model, where actin and myosin filaments are treated as charged cylinders, generates reasonable lattice spacings for skinned fibers. However, this model predicts excessive radial stiffness under osmotic pressure and cannot account for the initial pressure (∼1 kPa) required for significant compression. Good agreement with frog compression data is obtained with an extended model, in which S1 heads are weakly attached to actin when the lattice spacing is reduced below a critical value; further compression moves fixed negative charges on the heads closer to the myofilament backbone as they attach at a more acute angle to actin. The model predicts pH data in which the lattice shrinks as pH is lowered and protons bind to filaments. Electrostatic screening implies that the lattice shrinks with increasing ionic strength, but the observed expansion of the frog lattice at ionic strengths above 0.1 M with KCl might be explained if Cl⁻ binds to sites on the motor domain of S1. With myosin-myosin and actin-actin interactions, the predicted lattice spacing decreases slightly with sarcomere length, with a more rapid decrease when actin-myosin filament overlap is very small.     

X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths

Equatorial x-ray diffraction patterns from single skinned rabbit psoas fibers were studied at various ionic strengths to obtain structural information regarding cross-bridge formation in relaxed muscle fibers. At ionic strengths between 20 and 50 mM, the intensity of the 11 reflection, I11, of the relaxed state was close to that of the rigor state, whereas the intensity of the 10 reflection, I10, was approximately twice that of rigor reflection. Calculations by two-dimensional Fourier synthesis indicated that substantial extra mass was associated with the thin filaments under these conditions. With increasing ionic strength between 20 and 100 mM, I10 increased and I11 decreased in an approximately linear way, indicating net transfer of mass away from the thin filaments towards the thick filaments. These results provided evidence that cross-bridges were formed in a relaxed fiber at low ionic strengths, and that the number of cross-bridges decreased as ionic strength was raised. Above mu = 100 mM, I10 and I11 both decreased, indicating the onset of increasing disorder within the filament lattice.     

Ordering of the myofilament lattice in muscle fibers

The effect of pH on the muscle filament lattice in skinned rabbit psoas fibers was studied by X-ray diffraction. In relaxed fibers, the intensity of the 11 equatorial reflection, I11, remained constant between pH 7.0 and pH 6.0 and fell markedly when the pH was decreased to 5.5. The intensity of the 10 reflection was almost constant over this pH range. These results indicate that the thick-filament lattice is more stable than that of the thin filaments, and that the thin filaments are positioned within the thick-filament lattice by a charge-dependent force. In rigor fibers, the decrease in I11 over this pH range was much smaller, which shows that the thin filament lattice can also be stabilized by the presence of actomyosin crossbridges. These conclusions were confirmed by electron microscopy. Thus, the thin filaments can be positioned in the trigonal positions of the thick-filament lattice by two different mechanisms, one electrostatic and the other steric.     

X-ray equatorial analysis of crab striated muscle in the relaxed and rigor states

The structure of muscle projected along the fiber axis was studied by equatorial X-ray diffraction. The clectron-density distributions in axial projection of muscle were derived by the Fourier syntheses to a resolution of 7 nm in the relaxed and rigor states. The structure of the thick filament backbone (diameter about 21.5 nm) has a nearly smooth cylindrical surface and a low electron-density core (diameter about 7 nm) in the center. In the relaxed state, the center of gravity of the myoXXXin heads is situated at a radius of 19.6 nm from the center of the thick filament, lying just between the surface of the thick filament backbone and the surface of the thin filament (diameter about 8.4 nm). From the electron-density distributions in two slates. the amount of mass transfer from the thick filament to the thin filament was estimated. It was in accordance with that predicted from the structure derived bv the X-ray layer-line analyses.     

Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the thick filament (except near the tapered ends) is approximately 10 nm, or 16% of S2's total length, with an angular range encompassing 90 degrees axially and 120 degrees azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head-head interactions in IFM relaxed thick filaments.     

Donnan potentials from striated muscle liquid crystals. Lattice spacing dependence.

Electrochemical potentials were measured as a function of myofilament packing density in crayfish striated muscle. The A-band striations are supramolecular smectic B1 lattice assemblies of myosin filaments and the I-band striations are nematic liquid crystals of actin filaments. Both A- and I-bands generate potentials derived from the fixed charge that is associated with structural proteins. In the reported experiments, filament packing density was varied by osmotically reducing lattice volume. The electrochemical potentials were measured from the A- and I-bands in the relaxed condition over a range of lattice volumes. From the measurements of relative cross-sectional area, unit-cell volume (obtained by low-angle x-ray diffraction) and previously determined effective linear charge densities (Aldoroty, R.A., N.B. Garty, and E.W. April, 1985, Biophys. J., 47:89-96), Donnan potentials can be predicted for any amount of compression. In the relaxed condition, the predicted Donnan potentials correspond to the measured electrochemical potentials. In the rigor condition, however, a net increase in negative charge associated with the myosin filament is observed. The predictability of the data demonstrates the applicability of Donnan equilibrium theory to the measurement of electrochemical potentials from liquid-crystalline systems. Moreover, the relationship between filament spacing and the Donnan potential is consistent with the concept that surface charge provides the necessary electrostatic force to stabilize the myofilament lattice.     

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.     

Stiffness and fraction of Myosin motors responsible for active force in permeabilized muscle fibers from rabbit psoas

The stiffness of the single myosin motor (epsilon) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (T0) modulated by changing either [Ca2+] or temperature. The hs compliance is 43.0+/-0.8 nm MPa-1 in isometric contraction at saturating [Ca2+], whereas in rigor it is 28.2+/-1.1 nm MPa-1. The equivalent compliance of myofilaments is 21.0+/-3.3 nm MPa-1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5+/-1.7 kPa nm-1 in isometric contraction at saturating [Ca2+] (e0), and in rigor (er) it rises to 138.9+/-21.2 kPa nm-1. Epsilon, calculated from er and the lattice molecular dimensions, is 1.21+/-0.18 pN nm-1. epsilon estimated, using a thermodynamic approach, from the relation of T0 at saturating [Ca2+] versus the reciprocal of absolute temperature is 1.25+/-0.14 pN nm-1, similar to that estimated for fibers in rigor. Consequently, the ratio e0/er (0.33+/-0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e0 and er increase by approximately 40%. Epsilon becomes approximately 1.7 pN nm-1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.     

Dependence of thick filament structure in relaxed mammalian skeletal muscle on temperature and interfilament spacing

Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin filaments and myosin-containing thick filaments, but myosin-based regulation is unlikely to be preserved after thick filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the thick filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether thick filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the thick filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar filament shows that all zones of the filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on thick filament structure, disrupts the helical order of the myosin motors, and they move out from the filament backbone. Compression of the filament lattice of demembranated fibers by 5% Dextran, which restores interfilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the thick filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.     

Protein osmotic pressure and cross-bridge attachment determine the stiffness of thin filaments in muscle ex vivo

The properties of some models of the actin filament are compared with those of the thin filament in muscle. The greater stiffness of thin filaments ex vivo with respect to F-actin in vitro is attributed to the effect of both protein osmotic pressure and the attached cross-bridges. By comparing the stiffness of thin filaments in vitro and in isometric and rigor muscles the stiffness of thin filaments in relaxed muscle is computed. The upper limit of thin filament stretching is deduced to approach approximately 10 nm microm(-1). It is also calculated that, on stretching by 2.02 nm of the fully non-overlapped thin filament or by 1.59 nm of the thin filament on isometric contraction, the energy released on the hydrolysis of one molecule of ATP is fully used up.     

X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method.

Whole frog sartorius muscles can be chemically skinned in approximately 2 h by relaxing solutions containing 0.5% Triton X-100. The intensity and order of the X-ray diffraction pattern from living muscle is largely retained after such skinning, indicating good retention of native structure in fibrils and filaments. Best X-ray results were obtained using a solution with (mM): 75 K acetate; 5 Mg acetate; 5 ATP; 5 EGTA; 15 K phosphate, 2% PVP, pH 7.0. Equatorial X-ray patterns showed that myofibrils swell after detergent skinning, as also observed after mechanical skinning. This swelling could be reversed by adding high molecular weight colloids (PVP or dextran) to the extracting solution. By finding the colloid osmotic pressure needed to restore the in vivo interfilament spacing (3% PVP, 4 X 10(4) mol wt) the swelling pressure was estimated as 35 Torr in a standard KCl-based relaxing solution. The swelling pressure and the extent of swelling were less than acetate replaced chloride as the major anion. Detergent-skinned muscle lost the constant-volume relation between sarcomere length and lattice spacing seen in intact muscle. Changes in A band spacing were paralleled by changes in I and band-Z line spacing at a constant sarcomere length. After detergent skinning, I1,0 rose while I1,1 fell, a change in the relaxing direction. Since raising the calcium ion concentrations from pCa 9 to PCa 6.7 was without effect on equatorial or axial X-ray patterns, we concluded that these intensity changes were not due to calcium-dependent cross-bridge movement but rather to disordering of thin filaments in the A band.     

Radial forces within muscle fibers in rigor

Considering the widely accepted cross-bridge model of muscle contraction (Huxley. 1969. Science [Wash. D. C.]. 164:1356-1366), one would expect that attachment of angled cross-bridges would give rise to radial as well as longitudinal forces in the muscle fiber. These forces would tend, in most instances, to draw the myofilaments together and to cause the fiber to decrease in width. Using optical techniques, we have observed significant changes in the width of mechanically skinned frog muscle fibers when the fibers are put into rigor by deleting ATP from the bathing medium. Using a high molecular weight polymer polyvinylpyrrolidone (PVP-40; number average mol. wt. (Mn) = 40,000) in the bathing solution, we were able to estimate the magnitude of the radial forces by shrinking the relaxed fiber to the width observed with rigor induction. With rigor, fiber widths decreased up to approximately 10%, with shrinking being greater at shorter sarcomere spacing and at lower PVP concentrations. At higher PVP concentrations, some fibers actually swelled slightly. Radial pressures seen with rigor in 2 and 4% PVP ranged up to 8.9 x 10(3) N/m2. Upon rigor induction, fibers exerted a longitudinal force of approximately 1 x 10(5) N/m2 that was inhibited by high PVP concentrations (greater than or equal to 13%). In very high PVP concentrations (greater than or equal to 20%), fibers exerted an anomalous force, independent of ATP, which ranged up to 6 x 10(4) N/m2 at 60% PVP. Assuming that all the radial force is the result of cross- bridge attachment, we calculated that rigor cross-bridges exert a radial force of 0.2 x 1.2 x 10(-9) N per thick filament in sarcomeres near rest length. This force is of roughly the same order of magnitude as the longitudinal force per thick filament in rigor contraction or in maximal (calcium-activated) contraction of skinned fibers in ATP- containing solutions. Inasmuch as widths of fibers stretched well beyond overlap of thick and thin filaments decreased with rigor, other radially directed forces may be operating in parallel with cross-bridge forces.     

Unfixed cryosections of striated muscle to study dynamic molecular events.

The structures of the actin and myosin filaments of striated muscle have been studied extensively in the past by sectioning of fixed specimens. However, chemical fixation alters molecular details and prevents biochemically induced structural changes. To overcome these problems, we investigate here the potential of cryosectioning unfixed muscle. In cryosections of relaxed, unfixed specimens, individual myosin filaments displayed the characteristic helical organization of detached cross-bridges, but the filament lattice had disintegrated. To preserve both the filament lattice and the molecular structure of the filaments, we decided to section unfixed rigor muscle, stabilized by actomyosin cross-bridges. The best sections showed periodic, angled cross-bridges attached to actin and their Fourier transforms displayed layer lines similar to those in x-ray diffraction patterns of rigor muscle. To preserve relaxed filaments in their original lattice, unfixed sections of rigor muscle were picked up on a grid and relaxed before negative staining. The myosin and actin filaments showed the characteristic helical arrangements of detached cross-bridges and actin subunits, and Fourier transforms were similar to x-ray patterns of relaxed muscle. We conclude that the rigor structure of muscle and the ability of the filament lattice to undergo the rigor-relaxed transformation can be preserved in unfixed cryosections. In the future, it should be possible to carry out dynamic studies of active sacromeres by cryo-electron microscopy.