Conceived and designed the experiments: MCR RTT YG MR KAT. Performed the experiments: JL MCR RTT HS CL MR. Analyzed the data: SW. Contributed reagents/materials/analysis tools: SW HW CFA YG MR. Wrote the paper: SW MCR YG MR KAT. This author is deceased. This author is deceased.
Current address: Department of Pathology and Laboratory Medicine, The University of Texas - Houston Medical School, Houston, Texas, United States of America
Current address: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America
The authors have declared that no competing interests exist.
Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.
We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.
We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.
The conversion of the chemical energy of ATP into mechanical work by myosin involves coordinated changes in the actomyosin affinity and the orientation of myosin cross-bridges relative to the fiber axis
Differences in A-states and R-states predominately involve the configuration of a long cleft that divides the myosin MD into upper and lower 50 kDa subdomains, the so-called actin binding cleft. A-states, which have weak actin affinity, have an open cleft while R-states, which bind strongly to actin, have a closed cleft
The structural changes that occur in the A- to R-state transition are poorly defined, especially for myosin heads operating in situ. In the model described above, A-state myosin heads search for the myosin binding site on actin through a rapid equilibrium between attached and detached states until the MD alights on the myosin binding site on actin in the correct orientation for cleft closure. An alternative model involves diffusion of the myosin head on actin to the correct location and orientation for strong binding
Working strokes of 10–12 nm are about the maximum that can be achieved by a purely axial motion of the myosin lever arm and have been observed for single myosin S1 molecules
Visualizing active cross-bridges, including those bound to actin in the weak binding states thought to precede strongly bound force producing states, is essential for defining the structural transitions that constitute the working stroke of myosin. Because of their low actin affinity and possible heterogeneous structure when attached to actin, weakly-bound states are difficult to trap in vitro in numbers with sufficient homogeneity to be amenable to direct visualization by any of the powerful averaging techniques of cryoEM. However, 3-D visualization can be achieved using the technique of electron tomography (ET) which is capable of imaging individual molecules within a highly heterogeneous ensemble
IFM displays two levels of contraction depending on [Ca2+]. Stretch activation, which is characterized by rapid alternating contractions of antagonist muscles during flight, is the contraction mode most often studied
Active myosin heads interact with actin independently of each other so a snapshot of contracting muscle reveals the structure of multiple acto-myosin states within the context of the muscle lattice. Snapshots previously obtained from isometrically activated vertebrate striated muscle revealed a wide range of attachment angles in projections
Like the results obtained from vertebrate muscle, iso-HST cross-bridges visualized for the first time by ET also showed a wide range of attachment angles which could be ordered into a sequence compatible with a progressive 13 nm working stroke
Here we report a more detailed view of the rich variety of myosin head forms in the iso-HST state resulting from improvements in both data collection and analysis that have increased the resolution by 2.5× over the earlier work. The helical arrangement of actin subunits is now resolved, facilitating assignment of particular cross-bridge forms to specific actin subunits within the 38.7 nm repeat that spans from one Tn complex to the next. Multivariate data analysis (MDA) and classification of 3-D repeats is used to quantify individual cross-bridge forms from the number of repeats within each class
Advancements in data collection and analysis for ET since the iso-HST state was first reported
Structural analysis of active muscle is a challenge because of the presence of different structural and kinetic states of the myosin. The major analytical problem is the identification and grouping of self-similar structures so that averages with improved signal-to-noise ratio can be computed for comparison to much higher resolution structures obtained by crystallography and cryoelectron microscopy. The tomogram encompassed a myac layer, which is a 25–30 nm thick longitudinal section containing alternating myosin and actin filaments, ∼900 nm square containing 23 thin filaments and from which 515 repeat subvolumes containing a complete 38.7 nm axial repeat (hereafter referred to simply as repeats) were obtained. Our procedures used the thin filament centered on the target zone as a common frame of reference for alignment. To solve the problem of identifying groups of self-similar cross-bridge forms within the heterogeneous ensemble, we used MDA and classification of 3-D repeats and applied this procedure 12 separate times each focusing on separate critical regions of the structure. Averaged images obtained from the classification steps are referred to as class averages; those repeats that form the class are referred to as class members. Two applications of MDA focused on the left and right sides of the thin filament; averages derived from this we refer to as primary class averages. All of the structures in and around the target zone described in detail here were obtained from these two applications. Four more MDA applications identified cross-bridges in the region of Tn (dubbed “Tn-bridges”), four others were used to enumerate myosin head attachments on the eight actin subunits bracketing the target zone. Two more applications were used to verify the lever arm placements of primary class averages as well as to estimate the uncertainty in this critical parameter of cross-bridge structure. The approach is described in detail in a separate publication
Column averages in the previous work had an axial resolution of 12.9 nm which is insufficient to reveal the helix of F-actin subunits
Each frame is reassembled from left side, right side and Tn-bridge class averages and corresponds to one individual raw repeat whose number is in the upper right hand corner. Circled numbers indicate repeats that are shown at higher resolution in
The multiple different classification steps necessitated a reassembly procedure in which different class averages were combined to make a high signal-to-noise version of each raw repeat. Many different kinds and groupings of cross-bridges were found in the reassembled repeats (
We used MDA to separate the different structures, used the number of class members (raw 3-D repeats) to quantify the numbers of cross-bridges of a particular type and used quasiatomic model building to determine whether the interaction was strong or weak (this process is defined below). This data gives a frequency of forming a particular kind of cross-bridge on a specific F-actin subunit within the averaged repeat. The approach is not error free so to obtain some indication of its accuracy, we compared manual counts of cross-bridges with enumerations based on class membership
The distribution of actin-bound myosin heads is bimodal (
Weak attachments are shown on the left, and strong attachments on the right. The two actin long pitch strands are colored green and blue with the two target-zone actin subunits colored darker shades of green and blue. Occupancy on target-zone actins H–K was obtained from membership of primary class averages. Occupancy on actins R, S was determined from membership of Tn-bridge classification, while occupancy of actin D–G and L–O was determined from special classifications designed for these particular actins. Occupancy of target-zone actins H–K is plotted in darker shades of green and blue. Actin subunit designations correspond to the chain names in the coordinate files deposited in the Protein Data Bank, PDB – 2w49.
The remaining 22% of actin-bound myosin heads are spread over the ten non-target-zone actins for an average frequency of 8±4%, which is to say that 8% of the time these actin subunits have a myosin head bound in some manner. The distribution is not entirely flat but is slightly higher on the two actins on the M-ward side of the target zone (F & G) and on the four actins in the neighborhood of troponin (N, O, R, & S). The frequency of myosin heads bound on the two actins on the Z-ward side of the target zone (L & M) is only 2.6%. This strikingly low number differs by more than 2σ from the average of the other eight non-target-zone actins. The low number of heads on actins L and M is especially significant because it appears right next to the target zone, where both strong and weak myosin binding is highest. The average occupancy of the four troponin actins (N–Q) is 10.3±2.1% which is not significantly different from the average for all non-target-zone actins. Mostly, because of the low frequency of attachments to non-target-zone actins, D, E, L and M, the number of heads on the four actin subunits near troponin appears as a small peak in the distribution. Actins N and O are the location of rear bridges common in rigor muscle, so these actins can accept strong binding myosin interactions, but in iso-HST, surprisingly none were found.
From the number of total repeats, 515, we calculate a corresponding number of thick filament crowns, 458, and a total number of myosin heads potentially available, 3664. Our myosin head counts for all actin attached heads total 1948 heads for 53% attached to actin. The number of strongly bound heads is 1082 representing 29% of the total available. The proportion of strong binding cross-bridges as a fraction of the total available is consistent with measurements from vertebrate striated muscle during isometric contraction
We built into the global average of all repeats a single atomic model of the thin filament with 28/13 helical symmetry, containing 16 actin subunits, enough tropomyosin (TM) to cover the 16 actins, and four Tn complexes and then used that model for all the reassembled repeats (see
The location of the head-rod junction of the myosin heads is a particularly important parameter of the model fitting. It was verified by computing separate class averages based on features near the surface of the thick filament backbone, where the lever arms dominate. The raw repeat members of a primary class average were usually distributed over the membership of several thick filament class averages. Several independent fits of the lever arm could then be used to compute an axial and azimuthal standard deviation giving 12.6° or 1.5 nm for the axial orientation and 9° for the azimuthal angle
Identification of strong and weak binding myosin heads required an explicit criterion. We made this distinction based on the fitting of the MD into the class averages. Strong binding cross-bridges are those for which the MD fit the density without modification from the strong binding configuration, as defined by rigor acto-S1
We identify as
Although it was usually easy to tell whether or not the MD fit the density well without change from the starting structures, once the MD had to be moved the class averages lacked sufficient detail for unrestricted placement. We therefore adopted some guidelines for weak cross-bridge fitting. The entire starting structure was first moved as a single rigid body to get the closest MD fit possible and then the lever arm was adjusted. Lever arm adjustments were both axial and azimuthal. We permitted both azimuthal and axial MD translations and rotations, but usually azimuthal movements sufficed. There was insufficient definition in the MD density to require axial MD tilt to obtain a fit. The MD Cα backbone of weak binding bridges was not permitted to sterically clash with the TM backbone, which was in the high [Ca2+] closed position
Two other aspects of the model fitting are important. (1) The classification is not perfect and this affected some classes, in particular the decision as to whether a given cross-bridge is single- or double-headed. Assignment of a bridge class as single- or double-headed was made from examining the class members as described below. A consequence of this is that some single-headed cross-bridge classes contained variable numbers of second heads resulting in average bridge size at a constant contour threshold being larger than that expected for a single head. Likewise, some 2-headed bridge classes had variable numbers of single heads, causing the average 2-headed bridge size to be smaller than expected. (2) The model building was restricted to myosin heads attached to actin. The specimen also contains almost 50% myosin heads not attached to actin, which are located broadly. We expect these to average out, but there is always the possibility that variable amounts of density due to unattached heads will expand the class averages and not be fit by the atomic model.
We expected and found both strong and weak actin attachments in active contraction. There is little previously published information on the structure of weak actin attachments but strong binding attachments are well characterized from combinations of crystallography with cryoEM
We observed six kinds of cross-bridge configurations in the target zone (
The number in the upper right is the number of the corresponding raw repeat, NOT the number of raw repeats averaged within the class. Small panels to the left are the central section and an opaque isodensity surface view of the larger panel without the quasiatomic model. Actin long pitch strands are cyan and green with the target-zone actins in darker shades, TM is yellow and Tn orange. Strongly bound myosin heads are red, weak binding myosin heads are magenta, The essential light chain is dark blue and the regulatory light chain light blue. (A) shows a single headed cross-bridge on the left and a 2-headed, strong binding cross-bridge on the right. (B) shows a pair of 1-headed, strong-binding cross-bridges on actin subunits H and I. (C & D) have a 2-headed cross-bridge on the left and a 1-headed cross-bridge on the right, all strongly bound to actin. (E & F) are mask motifs with Tn-bridges. In (E) the right side M-ward weak binding cross-bridge is bound outside of the target zone to TM near actin subunit F while the one on the left is within the target zone on actin subunit I. In (F), the weak binding, left-side, M-ward cross-bridge is bound outside the target zone to TM near actin subunit G while the weak binding cross-bridge on the right is bound to target-zone actin subunit H. Tn-bridges have not been fit with a myosin head. These six reassembled repeats can also be viewed in Supporting
Mask motifs are a common structural feature in non-rigor IFM. iso-HST mask motifs were interpreted as consisting of a strong binding head pair on the Z-ward side and a weak binding head pair on the M-ward side
M-ward bridges of mask motifs are positioned on actin subunits F–I (
Some class averages appeared to be 2-headed, and when identified, were confirmed by examining galleries of the class members. If the majority of the class members were 2-headed, then the class was identified as 2-headed; if not, then it was identified as single headed. Although 2-headed cross-bridges are common in rigor muscle, they have not been quantified this accurately in active contraction. Two-headed bridges were only found in the target zone. Of the 1528 heads in the target zones, 442 of them (∼29%) were in 2-headed bridges. Of the 221 2-headed cross-bridges, 140 had both heads strongly attached (
The 2-headed cross-bridges of rigor usually have one rigor-like head with the second head's lever arm closer to 90° and this was true for one 2-headed cross-bridge class of active contraction, i.e. (
To determine the lever arm axial and azimuthal angles, we used heavy chain residues 707 and 840 in the Holmes et al. S1 structure, or the corresponding residues, 703 and 835, in the scallop transition state structure to define the lever arm axis. The angle between this vector and the thin filament axis defines the axial angle with angles <90° being rigor like and angles >90° being antirigor-like. In this convention, the axial lever arm angle of the Holmes S1 structure is 70.5° and of the scallop transition state structure 107°. When all strong binding heads are transformed to a single actin subunit, the lever arm positions sweep out an arc with an axial range of 77° and a distance of 12.9 nm at the S1–S2 junction (
(A) Ribbon diagrams are shown for only the heavy chains of all quasiatomic models (gold) and both starting myosin head structures (red and magenta) as docked onto actin in the strong binding configuration. (B) Plot of the axial angle vrs the azimuthal angle for the data shown in (A). Azimuthal angle measured looking M-line toward Z-line. (C) Plot of axial coordinate versus azimuthal angle for the same data. M-ward indicates the values obtained from myosin heads bound to the two actin subunits H and I at the M-ward end of the target zone; Z-ward indicates values obtained from myosin heads bound to Z-ward actin subunits J and K.
Values obtained after transforming myosin heads to a single actin subunit, I. Weak binding cross-bridges are aligned to the MD of the scallop transition state initial model. Vertical red and magenta lines indicate the position of the initial models within this coordinate frame. The positions of the lever arms for the starting atomic structures are shown as red and magenta vertical lines. (A) Distribution of lever arm tilt angles computed relative to the filament axis. Angles <90° are rigor-like and angles >90° are antirigor-like. The green curve is a Gaussian fit to the data with µ = 95.7°, σ = 19.8°. The fit for strong binding heads alone (not shown) is µ = 93.4°, σ = 19.5°. (B) Distribution of lever arm azimuths relative to the inter-filament axis. The inset gives the angular convention given a direction of view from M-line toward Z-disk. Red and magenta vertical lines show the azimuths of the starting atomic structures, which are very similar. Clearly, if starting-structure azimuth were the only influence, then the final azimuths in B should center around these vertical lines at 120°, as indeed the weak-binding target-zone bridges tend to do here. Direct Z-ward views of the unexpected azimuthal skewing observed for strong-binding bridges are shown in
We determined the azimuthal angle from the projection of the lever arm vector defined above, onto the equatorial plane of the filament lattice. A lever arm azimuth of 90° would be aligned parallel to the inter-thick-filament axis. The azimuthal angles thus defined are dependent on the actin subunit to which the myosin quasiatomic models are transformed, in this case subunit I, but the angular range is not. Surprisingly, the azimuthal range of all strong binding myosin heads is 126° (
The bimodal azimuthal angular distribution can be attributed to the 26° difference in azimuth presented by the myosin binding site on actin of the two target-zone subunits on each side of the thin filament as a consequence of its helical structure. When the starting structures are placed on the target-zone actins, their S1–S2 junctions are positioned clockwise from the line that connects the centers of the thick and thin filaments (
To provide a spatial reference, the models are displayed with the map of the global average. The horizontal dashed line represents the inter-thick-filament axis. All views are looking from the M-line toward the Z-line. (A) Scallop transition state starting model on actin subunits F–K. The S1–S2 junctions are positioned clockwise from the interfilament axis for all starting models on actins H–K. The S1–S2 junctions of starting models on F and G are located anticlockwise from the interfilament axis. However, no strong binding attachments occur on actins F and G. (B) Bridge models strongly bound to M-ward actin subunits H and I. The lever arms of the only two models that fall above the inter-thick-filament axis are bound to actin subunit H and have the appearance of early beginning-working-stroke conformations. (C) Bridge models strongly bound to Z-ward actin subunits J and K. (D) All strong binding models on their bound actin subunits showing azimuthal distribution skewed notably anti-clockwise from hypothetical dispersions centered around starting model positions in A.
Finally, the orientation of the “hook” of the myosin heavy chain in the starting structures (before rebuilding), which connects the myosin head to the S2 domain, is oriented away from the direction that the lever arm has to be bent to fit the strong binding bridges. This would suggest that the forces bending the lever arm azimuthally in situ, might also cause the lever arm to be twisted, a phenomenon which has been observed spectroscopically
Section compression, which in the present data reduced the inter-thick-filament spacing from 52 nm to 47 nm, may affect both the axial and azimuthal lever arm angles. We estimated the effect of section compression using a simplified model (
Left illustrates the initial state, prior to sectioning and section compression; right side illustrates the effect of section compression. Top row is the view looking down the filament axis; bottom row is the view looking perpendicular to the filament axis. Color scheme has the thick filament red, thin filament magenta, motor domain blue and lever arm black. The region of the target zone is colored cyan. We assume a worst case scenario, in which the thick and thin filament as well as the myosin motor domain are unaffected by compression and the entire effect is concentrated on the lever arm. Section compression decreases the interfilament spacing with a corresponding increase in section thickness. Widening of the section is assumed to be minimal since the reconstructions are scaled to the axial periodicities. See text for the values obtained from this model.
Many weak binding cross-bridge forms were identified in primary class averages, which revealed cross-bridges attached to actin subunits F–K. We divided weak binding bridges into two types, depending on the displacement of their MD center of mass from that of the strong binding attachments, and on whether they contact actin or TM (
Repeat # | # of Members | Actin Label | Type | Total Displacement (nm) |
298 | 22 | F | 2 | 4.94 |
343 | 26 | F | 2 | 5.49 |
311 | 15 | F | 2 | 5.97 |
343 | 33 | G | 2 | 4.55 |
348 | 24 | G | 2 | 4.29 |
126 | 19 | H | 2 | 4.30 |
107 | 24 | H | 2 | 2.76 |
117 | 26 | H | 1 | 1.52 |
348 | 27 | H | 1 | 0.97 |
246 | 46 | H | 1 | 0.73 |
356 | 27 | I | 1 | 2.63 |
73 | 24 | I | 1 | 0.96 |
311 | 22 | I | 1 | 0.70 |
126 | 32 | I | 1 | 0.44 |
246 | 18 | I | 1 | 0.19 |
117 | 26 | J | 1 | 2.49 |
118 | 16 | J | 1 | 1.75 |
356 | 21 | J | 1 | 1.67 |
46 | J | 1 | 1.39 | |
105 | 34 | J | 1 | 0.73 |
395 | 21 | J | 1 | 0.57 |
336 | 23 | K | 1 | 0.65 |
Repeat # refers to the index numbers identified in
# of members refers to the number of raw repeats present in the class.
Actin label refers to the actin subunit given in
Total displacement refers to the displacement of the MD center of mass relative to that of a strongly bound MD placed on actin subunit I. All models were transformed to actin subunit I prior to calculating the displacement.
*Presumed post rigor class average.
We transformed all weak binding cross-bridge quasiatomic models to align their MDs to the starting scallop S1 atomic structure placed on actin subunit I (
(A) Axial and azimuthal views of all weak binding cross-bridges aligned on the motor domain of the scallop transition state structure. This view illustrates the variations in lever arm compared with the starting scallop S1 structure. All weak binding bridges were built starting from the scallop transition state atomic structure, which is shown as a magenta colored ribbon diagram. Type 1 bridges are shown in gray and Type 2 bridges in gold, both rendered as chain traces. The single post-rigor conformation is colored light brown. (B) All weak binding cross-bridges superimposed on actin subunit I. This view illustrates the variations in MD position when referred to a single actin subunit. Coloring scheme is the same as for panel A. Note the relatively small axial dispersion of the Type 1 MDs compared to the broad dispersion of the Type 2 MDs.
Crossbridge Type | Number of classes | Axial Angle | Axial Displacement (nm) | Azimuthal Angle |
Scallop Transition State | - | 107° | +4.50 | 122° |
Holmes Rigor | - | 70.5° | −1.86 | 118° |
Type 1 | 13 | 92°–122° | +1.76–+6.87 | 89°–142° |
Type 1 |
1 | 53° | −3.8 | 117° |
Type 2 | 9 | 88°–93° | +1.03–+7.2 | 118°–121° |
All Weak Binding | 23 | 53°–122° | −3.8–+7.2 | 89°–142° |
When MD is aligned to a strong binding MD on actin subunit I.
*Presumed post rigor class average.
Axial displacement measured from the Z-coordinates of the Cα of heavy chain residue 835 of the scallop S1 structure. Axial angle and azimuthal angle measured from the coordinates of the Cα atoms of heavy chain residues 703 and 835.
The average of the axial angles excluding the post-rigor model, 105°±10°, is close to that of the starting scallop transition state model, 107°, and roughly evenly distributed around it. This axial range is less than twice the estimated uncertainty of the quasiatomic models and far less than the range for strong binding heads. Within the confidence limits of the model building, this range represents the inherent flexibility limits between the lever arm and the weakly bound MD.
Type 1 attachments are the most likely candidates for pre-working-stroke forms. When all Type 1 weak cross-bridges are transformed (aligned) onto actin subunit I, as opposed to a strong binding MD on actin subunit I, they suggest a progression in a
When all Type 2 weak binding bridges are aligned to actin subunit I, their MDs are all placed well beyond the azimuthal strong binding position on actin (
Here we combine modifications in both data collection and analysis to achieve richer and finer detail of the variety of myosin head forms in iso-HST than was possible previously
Previous work utilizing X-ray diffraction and ET concluded that 28–32% of the myosin heads are attached to the target zone for a frequency of ∼2.1 heads/target zone
The term target zone is defined as the segment of the thin filament where actin subunits are best oriented to form strong attachments to myosin heads projecting from adjacent, parallel thick filaments. Though defined initially for IFM
Previously, we inferred that the target zone was on average 2.5 actins long on each long pitch helical strand
We think that the highly restricted target zone cannot be due to the assumptions used to distinguish strong and weak binding attachments. Only Type 2 cross-bridges are found on M-ward actins F and G; even Type 1 attachments are not found on these actin subunits. Moreover, virtually no attachments of any kind were found on Z-ward actins L and M.
The relative orientation of actin subunits with respect to myosin head origins is the most obvious factor defining the target zone
Our sharply defined target zone implies a restrictive geometrical constraint on myosin head binding that is contradicted by the highly variable shape of strong binding cross-bridges, in particular their widely varying azimuthal lever arm orientations. If our two extremes for lever arm azimuth of strong binding myosin heads reflected intrinsic, state independent, myosin head flexibility, some heads should be able to bind strongly at all but two actins, D and E, while staying connected to the thick filament. Moreover, if actin azimuth alone were the limiting factor, we would expect a more gradual tapering of strong binding attachments from the center of the target zone. This we also do not see.
Type 1 weak binding attachments show a smaller azimuthal lever arm variation (
Based on the two initial crystal structures, we would expect that strong binding bridges would be found on actins F and G, but none are found. Even Type 1 weak binding bridges are not found on actins F and G. This suggests that an additional factor is limiting the target zone which may be the dynamic properties of TM. The Tn complex adds additional actin affinity to TM and holds it relatively securely at the ends of the actin repeat period but motion of TM would be expected to be highest midway between Tn complexes, exactly where the target zone is located. Reconstructions of actin-TM-Tn done by single particle methods revealed the weakest TM density midway between Tn complexes
Our results show that myosin head attachments occur all along the thin filament, although with much lower frequency than the target-zone attachments. Target-zone attachments account for 78% of all attachments but utilize only 28% of the actin subunits. The other 22% of attachments are distributed among the remaining 72% of actin subunits. Many of these attachments are non-specific with respect to the myosin binding site on actin. Some may represent collision complexes, but others, such as the Type 2 weak binding bridges on actin subunits F and G are both numerous and have a well defined appearance in the averages suggestive of some kind of specific interaction, even if novel. Outside of the target zone, actin subunits are labelled only 8% of the time on average (range of 3% to 13%). Nevertheless, these non-specific attachments occur with enhanced frequency on the 4 actin subunits near Tn and with strikingly low frequency on two actin subunits (L and M) adjacent to the Z-ward side of the target zone.
Initially, we thought actin subunits L and M never had myosin attachments, but a classification designed specifically for this region found some attachments, the density of which was generally poorly defined. Although it is possible that this represents statistical uncertainty, it may also indicate something special about these actin subunits. Non-specific attachments occur everywhere outside the target zone so the especially low frequency here may indicate an adaptation for IFM.
One in every seven subunits of IFM thin filaments is a ubiquinated form of actin named arthrin
Tregear et al.
(A) Data reproduced from Tregear et al. (2004) in which the target zone is assumed to be three actin subunits on one side and two actin subunits on the other. (B) Data from the present study, which includes only target-zone cross-bridges on two actin subunits from each side. Although the Tregear et al. data, which were measured by hand, have an overall Gaussian shape, the present measurements, which are based on quasiatomic model fitting, do not follow a strictly Gaussian distribution. The continuous line is a Gaussian fit with µ −0.86 nm and σ = 4.3 nm.
For comparison, we have replotted our fitting data shown in
Our results show that the number of actin-myosin attachments is relatively symmetric within the target zone, but is very asymmetric just outside the target zone, where the asymmetry in numbers is matched by an asymmetry in structure. The two actins M-ward of the target zone (F and G) are comparatively well occupied, while attachments to actins L and M on the Z-ward side of the target zone are rare. Weak attachments on actin subunits F and G (Type 2) differ the most from strong binding attachments, while those on actin subunit K (Type 1) have the smallest MD displacements from strong binding and are fewer in number.
Muscles are designed to generate tension while shortening and accomplish this by using filaments that are polar within the half sarcomere. Thus, the asymmetry we observe is not unexpected given the filament structures themselves. When a muscle shortens the target zone on the actin filament moves toward the M-line. On the M-line side of the target zone, on actin subunits F–H, we find the most unusual of the weak binding cross-bridge forms, Type 2. Although considered weak attachments, their structure is well defined in the averages and they are the only attachments found on those actin subunits. While not positioned on actin in a way that readily converts to strong binding, Type 2 attachments are placed so that if they remained stationary while the actin target zone moved M-ward by 1 or 2 actin subunits, they would be positioned to “bushwhack” the target-zone actins and form Type 1 weak attachments (Supporting
Conversely, if the muscle is stretched, which is to say the target zone moves Z-ward, there are literally no myosin heads positioned to bind the target zone. Adaptation to stretch would therefore require a different mechanism for rapidly placing more myosin heads on target-zone actins. It has been suggested that when a muscle is stretched, two-headed attachments increase by recruitment of second heads to single-headed bridges
The two types of weak binding attachments identified here may play differing roles in the recovery of tension after a quick release. Type 1 attachments are closest to the strong binding configuration and would thus appear to be best suited for contributing to Phase 2 rapid force recovery if the release distance was ∼5 nm
What exactly is holding the Type 2 bridges in place is not visible in the reconstructions. Their position is variable with respect to TM, both azimuthally and axially which argues against a specific interaction. One possibility is the N-terminal extension of the regulatory light chain. The sequence of this extension is known in
Based on column averages, it was concluded that the great majority of cross-bridge attachments were single-headed
Single headed myosin attachments are consistent with a variety of other structural evidence such as X-ray modelling of relaxed insect thick filaments
Previous analysis of iso-HST tomograms elucidated a working-stroke sequence by arranging all the quasiatomic models of cross-bridge fittings in a sequence based on the coordinate of the C-terminal residue at the S1–S2 junction
In the sequential ordering of structures from weak-to-strong binding
We characterized two types of weak attachments between myosin heads and the thin filament, with Type 1 attachments being closest in appearance to strong binding cross-bridges and with Type 2 being decidedly different. We think that Type 1 could be pre-working-stroke attachments because their MD position is closest to the strong binding configuration and they occur only within the target zone. Type 1 weak attachments are consistent with the idea that initial binding need not be precise
Our class averages at the moment lack the resolution and signal-to-noise ratio to be unambiguous but they suggest the following properties of the weak binding attachment that precedes strong binding. Type 1 cross-bridges can be arranged into a weak-to-strong binding sequence (
With the exception of one Type 1 weak binding cross-bridge, a possible post rigor conformation, all the weak binding forms on actin subunits F–K display a narrow range of axial and azimuthal lever arm orientations when aligned to the MD (
The fitting of strong binding cross-bridges generally required considerable axial alteration of the lever arm for both crystal structures, which differ by 36°/6.4 nm compared with the 77°/12.9 nm range observed here. Our observed range is consistent with previous estimates made by comparing crystal structures of myosin catalytic intermediates from different isoforms
The average axial lever arm angle obtained from a Gaussian fit to the data in
A consistent and surprising feature of the strong binding cross-bridges is the large, asymmetric distribution of the lever arm azimuths relative to the starting models. This is a feature of the data that has been extensively checked against different applications of MDA as well as against the original raw tomograms so we believe it to be real. The width of the distribution is affected by section compression, but even accounting for an additional 9° at either extreme, still gives a range of 110°. These azimuthal lever arm changes relative to the two initial structures gives the cross-bridges a more straightened appearance.
Cross-bridges with a straightened appearance have been observed previously in situ. They were first reported in EMs of IFM following AMPPNP treatment
Correlated changes in the actin layer line intensities with characteristic myosin reflections using an applied jump in temperature during isometric contraction
In vertebrate striated muscle, the intensity ratio of the 1,0 and 1,1 equatorial reflections is a measure of the change in myosin heads moving from thick filaments to thin filaments and these changes normally correlate with tension generation in isometric contractions
Accepting that our skewed azimuthal lever arm distribution is a property of active cross-bridges, the obvious question is what does it mean for myosin function in situ? Is it a reflection of intrinsic myosin head flexibility, is it an aspect of the weak-to-strong transition, or is it an aspect of the myosin working stroke? The broad lever arm azimuthal angular range seen for strong binding bridges is a conundrum. Placement of strong binding cross-bridges on actin can be limited only if the myosin head is given limited flexibility as suggested by the two crystal structures used for the fitting. Yet the changes observed in the strong (and weak) binding cross-bridges compared with the crystal structures imply substantial azimuthal flexibility that, if intrinsic to myosin heads, would permit strong binding bridges to form virtually anywhere along the actin 38.7 nm repeat. Even in rigor, where actin affinity might increase the target-zone size, cross-bridges are still largely confined to the target zone of contracting muscle. Intrinsic myosin head flexibility would seem to be an insufficient explanation for the strongly biased azimuthal change.
If the azimuthal lever arm distribution for strong binding cross-bridges were an aspect of the working stroke, we might expect to see a relationship between axial angle, representing progress through the working stroke, with increasing, or decreasing azimuthal angle. However, graphs of axial tilt angle versus azimuthal angle for the strong binding cross-bridges fail to show the obvious correlation (
That there is no coupling between axial and azimuthal lever arm angles may be an effect of S2. Cross-bridges do not emerge from the thick filament backbone at the S1–S2 junction; they originate where S2 emerges from the thick filament backbone. S2 is widely thought to provide a flexible tether that can bend radially, as well as azimuthally around the thick filament surface, to facilitate actin-myosin attachment. If S2 swings azimuthally during cross-bridge attachment so that it becomes angled with respect to the filament axis when force is initiated
In iso-HST, we do not observe where S2 emerges from the thick filament backbone, we only see the S1–S2 junction of the lever arm and so cannot directly evaluate this effect. Where S2 has been observed in swollen IFM fibers, the range of directions suggests that S2 swings equally well both clockwise and anticlockwise, about the thick filament surface
We can think of two mechanisms that would bias the azimuthal bend of the lever arm to produce a cross-bridge that appears azimuthally straightened in comparison to the crystal structures. One of these involves the weak-to-strong transition, the other invokes an active azimuthal component to the working stroke. Either mechanism would enable the S1–S2 junction to be positioned anticlockwise of the inter-filament axis as depicted in
(A–C) Conversion from weak-to-strong binding according to Scenario 1. (D–F) Active azimuthal component to the working stroke. View direction is M-line toward Z-line. Myosin is colored either red (strong binding) or magenta (weak binding). Actin subunits are green and blue. Three successive levels of S2 origins are shown in shades of brown that darken with distance from the observer. The lever arm is the line originating on the red (or magenta) MD while S2 is shown as a short segment when oriented nearly parallel with the filament axis and becomes longer when angled with respect to the filament axis. The horizontal line is the inter-filament axis. Arrows show the direction of the torques (not their magnitude) produced during the weak-to-strong transition or as a component of force generation and filament sliding. The direction of thin filament movement during sarcomere shortening is toward the observer. (A & D) Initial weak binding is shown, which in (A) begins away from the strong binding orientation and in (D) begins in the strong binding orientation but with actin binding cleft open. (B) Conversion to strong binding involves diffusion of the MD clockwise on actin which swings the S2 anticlockwise about the thick filament. (C) Force production realigns S2 with the filament axis while bending the lever arm azimuthally. (E) Transition from weak to strong binding involves no change in myosin orientation on actin, just a closing of the actin binding cleft. The lever arm in (D & E) is in the same orientation suggested by the crystal structures. (F) An azimuthal component to the working stroke moves the lever arm clockwise around the thick filament. This figure can be seen as an animated sequence in Supporting File S1.
The range of structures of Type 1 cross-bridges (
In the first scenario (Supporting
In the second scenario (Supporting
The first scenario is supported by the two strong binding structures which fell above the inter-filament axis in
The first scenario bears some resemblance to the “roll and lock” mechanism described by Ferenczi et al.
Several observations made with in vitro motility assays have shown a torque component to the working stroke, dubbed “twirling”, inferred from rotations of the actin filament in gliding assays
The azimuthal movement of the lever arm under this mechanism would not only straighten the cross-bridge but would also impose a clockwise torque on both the thick and thin filament, opposite the one imposed by the weak-to-strong transition. If filament sliding was possible, the applied torque would impose a left handed rotation to the thin filament, matching the observation in vitro
Generally, motility assays are performed with the motor bound to a substrate via the rod domain (S2 plus LMM), which would argue that the S1–S2 junction is comparatively immobile and the actin filament free to move during these assays. If the actin filament were fixed as it appears to be in IFM and the S1–S2 junction free to move on its S2 tether, then the same conformational change in the myosin head that causes left hand twirling of free F-actin would rotate the lever arm anticlockwise or right handed with respect to the thin filament to produce myosin head straightening as observed.
Beausang et al.
Forces or components of forces that alter the lever arm azimuth must also impose torsional forces on the thick and thin filaments. Neither the thin filaments, anchored at the Z-disk nor the thick filaments, which are bipolar, can rotate as rigid bodies; they can only change their helical twist in response to an applied torque. For the thin filaments this is not as absolute as for thick filaments since it depends on the compliance of actin crosslinks in the Z-disk. The rotation of the MD on actin during the weak-to-strong transition produces an anticlockwise torque (looking Z-wards) on both the thick and thin filaments (
There is no evidence in the present tomograms or in any published data from IFM of a change in the half pitch of the thin filament which would be a necessary consequence of any torsional rotations in situ. The thin filament azimuth in our tomograms of active contraction appears virtually identical to that found in other states of IFM investigated by electron microscopy alone
There is some evidence from X-ray diffraction of helical changes in the thin filament of vertebrate striated muscle during isometric contraction
Perhaps the lack of visible effects on the helical structure of the filaments is due to the differing sense of the two hypothesized torques; the weak to strong transition produces a clockwise torque, twirling results from an anticlockwise torque. More likely the thick and thin filaments are too stiff to be significantly altered by these forces and most of the torsional force is dissipated by S2 and the myosin lever arm.
We have shown multiple myosin head structures in isometrically contracting muscle consistent with both weak binding and force producing cross-bridges. We can infer from these structures that the weak to strong transition involves largely azimuthal movements of the myosin MD and consequent changes in the lever arm. This agrees with X-ray diffraction observations of the weak to strong transition in which changes in intensity occur largely on the 38.7 nm actin layer line which is sensitive to increases in stereospecific binding, on the equatorial reflections which are sensitive to azimuthal and radial changes in structure and with minimal changes in the 14.5 nm meridional reflection, which is sensitive to lever arm or MD axial orientation
Rapid freezing with simultaneous monitoring of fiber tension and stiffness was performed on a Heuser Cryopress freezing head
Details of all the data analysis procedures have been described
Repeats spaced 38.7 nm apart axially and containing a 60.7 nm axial length of the actin filaments, their bound cross-bridges and adjacent thick filament segments were centered on the actin target zones. Alignment error between the raw repeats was minimized by choosing as the reference the single large structure, the thin filament, which was in common to all repeats. A global average of all extracted raw repeats was used as the initial reference and was followed by MDA and multireference alignment. After several cycles of multireference alignment, the final alignment used a single reference as it was desirable to fit one atomic model to the thin filament for all class averages and raw repeats that would be used for all subsequent model building.
The purpose of MDA is to sort the highly variable cross-bridge forms within the repeats into self-similar groups. A key part of MDA is the generation of Boolean masks, which select a set of contiguous voxels defining a region of the repeat within which patterns of density will be identified. To retain the greatest variation in structure possible, we generated several classification masks. Two of these selected myosin heads bound to either the left or right side of the actin target zones; these are the primary class averages. Four masks were used for the troponin region, four masks were specific for actins outside of the target zone and two were used for the surface of the thick filament to verify the lever arm positions. Class averages from each classification were subsequently reassembled to make composite class averages
Because of the complex manner in which the individual repeats are reassembled, conventional methods of resolution determination, such as the Fourier Shell Correlation are meaningless. However, a qualitative measure of the resolution can be obtained by the fact that the helix of actin subunits can be observed, which requires at least 5.5 nm resolution. The resolution of the previous work was limited to 12. 9 nm so the improvement is at least a factor of 2–3.
Quasiatomic models were built in an hierarchical fashion with modifications from earlier studies using rigor IFM fibers
For cross-bridges whose lever arm orientations were angled toward the rigor configuration, we used an atomic model adapted from the Holmes et al. rigor acto/S1 complex
Models were built separately into each left-side target-zone average and each right side target-zone average giving 20 atomic models for each. These were then combined as necessary to produce all of the complete quasiatomic models. This was also done for the troponin bridges. The position of the head-rod junction of the cross-bridges was checked in two ways, either against the density of the raw repeat subvolume or against a column average of the thick filament surface classification. Adjustments were made if indicated.
Figures and movies were constructed using CHIMERA
Expanded summary of weak attachment models fitted in primary mask class averages.
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This is a movie of the quasi atomic model shown in
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This is a movie of the quasi atomic model shown in
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Axial diffusion of Type 2 weak binding bridges. This movie shows Z-ward diffusion of a Type 2 weak binding myosin head as might occur during sarcomere shortening when the target zone moves M-ward. The myosin heavy chain is colored magenta to indicate a weak binding state. The myosin head starts out attached to TM in the region of actin subunit F. It then diffuses Z-ward until reaching actin subunit J at which point it is now positioned as a Type 1 weak binding myosin head and can begin the weak-to-strong transition, which largely involves azimuthal movements. When strong binding occurs, signified by change of color to red, the lever arm then moves Z-ward to complete the power stroke. Morphing done using Chimera.
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This movie illustrates how S2 can affect the conformation of a myosin head during the weak-to-strong transition followed by a working stroke. An 11 nm long segment of coiled-coil has been attached at the S1–S2 junction. The S2 segment is assumed to be compliant but its origin at the myosin filament backbone is considered fixed; the myosin head is assumed to be non-compliant. The myosin head is initially bound weakly (signified by the magenta colored myosin heavy chain). The weak-to-strong transition involves largely azimuthal movements on its actin subunit (subunit K in this case). When strong binding occurs, signified by the change in heavy chain color to red, the S2 is angled which will result in an azimuthal component to the working stroke.
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This movie illustrates a second way that S2 can affect the conformation of a myosin head during the weak-to-strong transition followed by a working stroke. Similar to
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This powerpoint file contains the panels of
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