Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths

Figures

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  Figure A1. (a) Geometry of the diffraction pattern looking down the meridian. The two circles represent the sizes of two meridional peaks of different width across the meridian. The horizontal lines represent the thickness of the Ewald sphere relating to an X-ray beam where the wavelength spread can be represented by a thickness t; (b) Graphs showing the true volume V and the estimated volume V* as a function of Ewald sphere thickness t; (c) Graph showing the %age error relative to Ewald sphere thickness t, of multiplying the meridional peak area by the full width at half maximum of the peaks (see Table 2).

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  Figure 1. (a) Electron density map (low resolution) of F-actin labelled with myosin S1 fragments, with no ATP present based on electron microscopy and 3D reconstruction; (b) Representation of the myosin head with the lever arm up as in relaxed muscle and down as in normal rigor (a), assuming a vertical actin filament on the right of the motor domain.

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  Figure 2. (a) Left half of the X-ray diffraction pattern from relaxed Turbot fin muscle, 8 h exposure time. Myosin layer-lines (orders of 429.6 Å) are labelled ML1 to ML6 and meridionals M4 to M15; (b) Right half of the X-ray diffraction pattern from the short SL rigor Turbot fin muscle, 8 h exposure time. Actin layer-lines are labelled AL1 to AL13. The meridian is vertical, the equator horizontal. The lower strip shows the intensity scale used.

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  Figure 3. (a) Left half of the X-ray diffraction pattern from relaxed Turbot fin muscle, 8 h exposure time. Myosin layer-lines (orders of 429.6 Å) are labelled ML1 to ML6 and meridionals M4 to M15; (b) Right half of the X-ray diffraction pattern from the longer (2.5 µm) SL rigor Turbot fin muscle, 8 h exposure time. Actin layer-lines are labelled AL1 to AL13. The meridian is vertical, the equator horizontal. The intensity scale is shown at the bottom.

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  Figure 4. (a) The difference pattern relaxed minus 2.2 µm rigor; (b) The difference pattern relaxed minus 2.5 µm rigor; (c) The difference pattern of the short SL rigor pattern minus the longer SL rigor pattern. All from Turbot fin muscle, 8 h exposure. Actin layer-lines are labelled and the beating actin myosin layer-line is labelled AM+1 (Koubassova and Tsaturyan [12]). The positive and negative differences are coded as in the lower strip for all patterns; reds, purple, and black show increases, light orange and yellows show decreases, dark orange shows no change.

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  Figure 5. (a) Positions and widths of the horizontally integrated profiles taken parallel to the meridian of the X-ray patterns from the three muscle states: relaxed, short SL rigor, and longer SL rigor; (b) Positions and widths of the vertically integrated profiles taken along the actin layer-lines in the X-ray diffraction patterns from the three muscle states. The traces along these regions are shown in Figure 6 and Figure 7.

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  Figure 6. Integrated intensity profiles in the strips indicated in Figure 5a, which are all parallel to the meridian of the diffractions patterns. Layer lines referred to in the text are labelled. Trace colours: relaxed state (green), short rigor (blue), and longer rigor (red). (a) Meridional profile integrated out to 0.002123 Å−1 on either side of the meridian; (b) Profile parallel to the meridian (axial profile) centred on the peak of the 6th actin layer-line horizontally integrated from 0.003663 to 0.01428 Å−1; (c) Axial profile horizontally integrated from 0.01428 to 0.01959 Å−1; (d) Axial profile horizontally integrated from 0.01959 to 0.02490 Å−1; (e) Axial profile horizontally integrated from 0.02490 to 0.03021 Å−1.

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  Figure 7. Vertically integrated horizontal profiles taken from the X-ray diffraction patterns from Turbot fin muscle in the relaxed state (green) and put into rigor at short (blue) and longer SL (red). The profiles are 0.00210 Å−1 wide and are centred on actin layer-lines 1 to 7 (a–g) as indicated.

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  Figure 8. (a) Difference map created by subtracting the synchrotron X-ray diffraction pattern of Plaice fin muscle put into rigor at SL = 2.5 µm from the diffraction pattern of Plaice fin muscle put into rigor at SL = 2.2 µm. The actin and myosin layer-lines are labelled AL1 to AL2 and ML1 to ML3 respectively, the myosin meridionals M2 to M3 and the beating actin myosin layer line AM−1. An attenuation strip was used to reduce the total intensity of the equatorials; (b) Horizontally integrated profiles of the meridian in the two summed X-ray diffraction patterns from Plaice fin muscle put into rigor at two different sarcomere lengths 2.2 µm (blue trace) and 2.5 µm (red trace). The meridional reflections are labelled M1 to M3. The fine sampling on the meridian will change with sarcomere length due to interference between the two halves of the A-band [7].

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  Figure 9. Equatorial profiles from the summed X-ray diffraction patterns of Plaice fin muscle put into rigor at SL = 2.2 µm (blue trace) and 2.5 µm (red trace), integrated from 0 to 0.00043 Å−1 on either side of the equator. The positions of the six equatorial reflections (including Z-line peaks) present in these profiles are labelled.

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  Figure 10. Electron density maps produced using the average amplitudes from the first five equatorial A-band reflections from Plaice fin muscle put into rigor at SL = 2.2 µm (a,c) and 2.5 µm (b,d); (a,b) were computed using phase set 1 (0°, 0°, 180°, 0°, 0°) and (c,d) were computed using phase set 2 (0°, 0°, 180°, 180°, 0°). A few unit cells of the A-band lattice are shown in each map and the actin and myosin filament positions are labelled A and M respectively. The colour scale used throughout is shown at the bottom. Densities were scaled to give the same total mass in each case.

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  Figure 11. (a) The left half of a simulated diffraction pattern of a helix with the symmetry of the actin filament (a 13/6 helix of repeat 360 Å and subunit translation 27.36 Å) obtained with the program Helix [30]. Intensity from AL1 upwards gradually moves away from the meridian up to AL3 and then moves back towards the meridian up to AL6. Something similar then occurs from AL7 to AL13 which is on the meridian; (b) The right-hand half of part of the low-angle diffraction pattern from the full overlap rigor muscle (cf. Figure 2), with the same gernal shape of diffraction pattern, but with peaks closer to the meridian than is expected from just actin (a). For further details see text.