The corrected layer line intensities and the thick myosin filament model.

<p>(A) Comparison of the corrected layer line intensities (gray curves) from nearly full-overlap muscles and the intensities calculated from the optimum model with the azimuthal perturbations and C-proteins (the full-filament overlap model) (black curves). The intensities indicated by dashed lines in the region of <i>R</i><0.0254 nm⁻¹ are not used for modeling (see text). The intensities are normalized as in Fig. 4B. (B) Part of the radial projection of the crossbridge helices of a myosin filament in the perturbed region showing the azimuthal locations of crowns 1, 2, 3 and 4 and the displacements due to the axial and azimuthal perturbations. The abscissa axis is denoted by <i>φ</i> instead of 0∼2π<i>r</i>. Blue filled circles and black open circles denote respectively the shifted and regular locations of the crossbridge centers along the three 9/1 helical lines. Each dashed line shows each 9/1 helix in the three-stranded regular helical filament. (C) and (D) The full-filament overlap models in projection view in the regular (C) and perturbed (D) regions. (E) and (F) Those in cross-sectional view at the axial level of crown 2 in the regular (E) and perturbed (F) regions. (G) and (H) Those in side view in the regular (G) and perturbed (H) regions within the basic repeat. (I) and (J) Close-up views of the inter- and intra-molecular interactions between the myosin heads at the asterisk in (G) and (H). Yellow spheres mark the converter portion of the red head. The bottom of (G)-(J) is toward the M-line. The backbone is described as a structure-less cylinder. Scale bar, 10 nm.</p

Modeling crossbridge structure of the frog skeletal thick myosin filament.

<p>(A) Distributions of the perturbed region (blue), the regular region (green) of myosin crossbridge arrays and the C-protein region (red) along the thick filament in resting frog muscles. (B) Overview of the thick filament model including C-proteins. Members of paired heads constituting a single myosin crossbridge are distinguished by a red or purple color. C-protein is bound to the thick filament backbone every at the level of crown 1. Eleven domains of a C-protein having a molecular weight of ∼130 kDa are shown by white spheres, each having a diameter of 4 nm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone.0052421-Luther1" target="_blank">[18]</a>. The backbone of the myosin filament is shown as a gray cylinder. Upper, a top view and lower, a side view. (C) Parameters describing the arrangement of a two-headed myosin crossbridge in terms of a 68-sphere model on the filament backbone in ADP and Pi-bound state. The <i>z</i>-axis is parallel to the filament axis. <i>φ</i> is the rotation angle of crossbridges about the filament axis and <i>ε</i> is the rotation angle of the two heads of a crossbridge about the <i>z-</i>axis. <i>r_h</i> is the average of helical radii of myosin crossbridges. Paired heads of a single crossbridge and the backbone are denoted as in (B).</p

Axial intensity profiles of the C-protein-associated meridional reflections.

<p>The x-ray diffraction pattern was obtained at the Advanced Photon Source. (A) Those of the C1/M1 region. (B–E) Those of the clusters of the C2 to C5 meridional reflections. The experimental intensity profiles are denoted by red dotted curves and the fitted Gaussian functions are shown by black curves. (F) Axial spacings of each C-protein and each myosin reflection when divided by their reflection index. They are shown by blue and red full circles, respectively. Bars on the circles are the standard deviation of the mean from four data sets. The average period of C-protein is 45.33±0.58 nm and that of myosin is 42.96±0.11 nm. In (F), the spacing (44.5 nm) of the reflection which was enhanced by labeling antibody to C-protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone.0052421-Rome1" target="_blank">[40]</a> is shown by the blue open circle. In (A), there are triplet of reflections in 0.0205 nm⁻¹< <i>Z</i><0.0245 nm⁻¹ with a main peak at 1/44.5 nm⁻¹, and the axial positions of the reflections assigned as C1 and M1 are depicted by vertical lines. TN1 denotes the sampled first order troponin-based reflection.</p

The cylindrically-averaged difference Patterson function and the corrected layer line intensities.

<p>(A) The <i>r</i>-<i>z</i> map of the cylindrically-averaged difference Patterson function [Δ<i>Q</i>(<i>r, z</i>)] calculated from the observed M1 to M11 layer line intensities. The map is contoured at levels between 0 and 410 at the intervals of 4.1. Only positive peaks are shown and negative peaks are omitted. The borderline (a red curve) is drawn distinguishing the region of inter-crossbridge vectors within a single filament from that of inter-filament crossbridge vectors. (B) Comparisons of the intensities (black curves) corrected by the cut-off method and the original layer line intensities from full-overlap muscles (gray curves). The layer line intensities are normalized so that the sum of the intensities except for the region close to the meridian (<i>R</i><0.0254 nm⁻¹) is identical between the corrected and original intensities (see text).</p

Summary of parameters of the optimum myosin filament models.

<p>Parameters used to define the azimuthal orientation of myosin crossbridges on the thick filament in the model calculation. The first column identifies the parameters used in the modeling; <i>ε</i>, the rotation angle of a head about the <i>z</i>-axis and <i>r_h</i>, the helical radius of a head where subscripts (<i>r</i>, <i>p</i>) denote those for the regular and perturbed regions, respectively. <i>φ</i>, “is the” azimuthal rotation angle of a myosin crossbridge in the perturbed region where subscripts (1, 2, 3) correspond to crown levels 1, 2 and 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone-0052421-g006" target="_blank">Figs. 6B and H</a>. The second and third columns identify the parameter values for the full-filament overlap and non-filament overlap models, respectively. The paired heads of a single myosin crossbridge are denoted as a red head and a purple head.</p

X-ray diffraction patterns from resting frog skeletal muscles.

<p>(A) An x-ray diffraction pattern in the resting state of live frog skeletal muscles with full thick-thin filament overlap, obtained at the Photon Factory. The background intensity is subtracted. The fiber axis is vertical. M1 to M11 denote the first to eleventh order myosin-based layer lines with a basic period of 42.9 nm. C2 to C5 denote the clusters of meridional reflections that may be assigned as the second to fifth order C-protein-associated meridional reflections with a period of 45.3 nm (see text). The values in parentheses are the axial spacing in real space (in nm). M, the meridional axis and E, the equatorial axis. (B) The observed intensity distributions on the M1 to M11 layer lines.</p

Inter- and intra-molecular interactions between heads of myosin crossbridges.

<p>The directions of the view are same as in Figs. 6I and J. The converter domain (residues 724–764) is marked by a yellow color. The secondary structure of the myosin heavy chain is shown as in Fig. 1A. (A) The inter-molecular head-head interactions in the regular region. (B) An atomic structure model of a tarantula myosin crossbridge (PDBID: 3DTP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone.0052421-Alamo1" target="_blank">[5]</a> without the S2 structure and by its 75-sphere model made in the same way as in the case of the frog sphere model. A head (red) in tarantula myosin is put with the same orientation of the overall structure onto a myosin head (red) in the regular region of the full-filament overlap model. In both (A) and (B), the blocking head is toward the converter domain of the adjacent head. (C) The inter-molecular interactions and (D) the intra-molecular interactions between heads in the perturbed region. (E) - (G) Close-up views of electrostatic potential on the molecular surface of the myosin heads in the inter-molecular interactions of the regular region in (E) and those in the inter- and intra-molecular interactions of the perturbed region in (F) and (G), respectively. The negatively and positively charged regions are shown by red and blue colors, respectively, in the range of −1∼1 k_bT/e (k_b, Boltzmann constant; T (310K), absolute temperature; e, electron charge). For the charge distribution, one of myosin heads is diagrammed as a ribbon-and-wire cartoon and the other head is shown as a molecular surface.</p

A frog skeletal myosin filament model using the homology model of S1.

<p>(A) and (B) Optimum models of the homology model of S1 in projection view down the filament axis in the regular and perturbed regions, respectively. The optimization was done in the same way as described in the text. (C) and (D) Those in cross-sectional view at the axial level of crown 2 in the regular and perturbed regions, respectively. (E) and (F) Those in side view within the basic repeat in the regular and perturbed regions, respectively. (G) and (H) Close-up views of the inter- and intra-molecular interactions between the myosin heads at the asterisk in (E) and (F), respectively. Yellow spheres mark the converter portion of one head. The bottom of (E) – (H) is toward the M-line. The filament backbone is described as a structure-less cylinder. Scale bar, 10 nm. The orientations of myosin heads in the homology model are very similar to those of the S1.ADP.Pi model in the text (Figs. 6C–J). The <i>RDI</i> of this optimum model is ∼0.32, slightly higher than that of the full-filament overlap model.</p

An atomic structure model of a myosin II S1 made by a homology modeling.

<p>(A) The atomic structures of myosin II subfragment 1 (S1) in ADP.Pi-bound state (yellow) made by a homology modeling method (cf. Fig. 1A) (see text). S1 molecules in nucleotide-free and ADP.Pi-bound states are shown by green and gray colors, respectively. The radius of gyration of S1 (C°-model) is ∼4.66 nm in the homology model. Each part of the motor domains of three S1 molecules was superimposed on each other, where the models shown by green and grey colors are the same as in Fig. 1A. The secondary structure of the myosin heavy chain is shown as in Fig. 1A. (B) A Ramachandran diagram of S1 in the homology model. The diagram was generated using the CCP4 tool, rampage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone.0052421-Lovell1" target="_blank">[36]</a>. The ratios of the number of residues to the total number in favored regions, allowed regions and other (outlier) regions are 78.1%, 18.5% and 3.4%, respectively.</p

Atomic structure modeling of a myosin II S1.

<p>(A) Comparison of the atomic structures of myosin II subfragment 1 (S1) in nucleotide-free and ADP.Pi-bound states. Atomic structures of myosin II S1 in nucleotide-free state (green) and in ADP.Pi-bound (ADP.Pi) state (gray). The structure in nucleotide-free state was taken from PDBID: 2MYS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone.0052421-Rayment1" target="_blank">[31]</a>. The part of the motor domains of the S1 molecules is superimposed on each other. The secondary structure of the myosin heavy chain is diagrammed as a ribbon-and-wire cartoon. The radius of gyration of S1 (C°-model) is ∼4.56 nm in the nucleotide-free model and ∼4.45 nm in the ADP.Pi-bound model. (B) A Ramachandran diagram of S1 in the ADP.Pi-bound model. The diagram was generated using the CCP4 tool, rampage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone.0052421-Lovell1" target="_blank">[36]</a>. The ratios of the number of residues to the total number in favored regions, allowed regions and other (outlier) regions are 77.2%, 19.4% and 3.4%, respectively.</p