Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

15A/2005G310

Orientations of Resting Myosin Crossbridges from Skeletal Muscles Determinedby X-ray Fiber Diffraction
Kanji OSHIMA, Yasunori TAKEZAWA, Yasunobu SUGIMOTO, and Katsuzo WAKABAYASHI*
Division of Biophysical Engineering, School of Engineering Science, Osaka University,Toyonaka, Osaka 560-8531, Japan
Introduction
The orientation of a crossbridge around the fiber axis
was not determined from the intensity analysis of themeridional reflections which was reported in the previous
reports [1,2,3,4] because the projected density of acrossbridge onto the fiber axis is identical even if theorientation of a crossbridge is different around the fiberaxis. Therefore we used the intensities of the layer-linereflections of diffraction patterns from muscles at fulloverlap length and those at non-overlap filament length in
the relaxed state to determine the orientation ofcrossbridge by rotating independently each head of twoheaded crossbridges around the fiber axis.
Experimental
Live frog sartorius muscles were used for X-ray studies.
X-ray diffraction experiments were performed at BL15A1.
The 2D-X-ray diffraction patterns from muscles wererecorded with an image plate at the specimen-to-detector
distance of ca. 2.4 m. Whether muscles were stretched to
the non-overlap length was made by measuring thediffraction periods from the sarcomere of overstretchedmuscles by diffraction of a laser light.

∆Q(r,z) map to recalculate the intensity data of the layerline reflections from a thick filament without thesampling effect.
The recalculated intensity data of muscles at full andnon-overlap lengths were very similar to each other. This
indicates that the sampling effect was mostly removed
and the intensities of a single myosin filament wereobtained. The comparison between the obtained ∆Q(r,z)
with the theoretical Q(r,z) of a three-stranded 9/1 helix oftwo-headed crossbridges showed that the radial positionof a crossbridge along a helix was ~12.6 nm from thepeak around (z, r)=(0, ~22 nm) and that the two heads ofa crossbridge were separated axially. We employed suchinformation for a crossbridge arrangement to carry out theintensity calculation of the layer-line reflections by theFourier-Bessel transformation.
In the best-fit model, each myosin head of acrossbridge has also a different orientation around thefiber axis. The distal ends of two heads of a crossbridge
appear to orient in the same direction when seeing from a
top view, making a U-shape structure according to theconfiguration of the myosin molecule (Fig. 1B).

Results and Discussion
The myosin-based layer-lines are partially sampled by
the hexagonal filament array. In order to remove thesampling effect from the layer-lines, a cylindricallyaveraged difference-Patterson function ∆Q(r,z) was used.
The ∆Q(r,z) of a myosin filament was constructed byusing the intensity data from the first to the sixth order
myosin-based layer-line reflections in a radial range of R-1< 0.157 nm using the equation of(1)where Il(R) is the intensity distribution along the lthlayer-line reflection of the diffraction pattern and c is thecrystallographic period (43.02 nm) and the J0(2πγR) is thezeroth order Bessel function of an argument of 2πγR (Fig.
1A). In Fig.1A, positive peaks appeared clearly on the∆Q(r,z) map, corresponding to the vectors between thecenters of gravity of crossbridges on a three-stranded
helical arrangement. Some weak positive peaks whichappeared in the region of r > ~32 nm were interpreted as
the inter-filament vectors among thick filaments whichcame from the lattice sampling. Therefore we removed
these peaks (outside the red curve in Fig. 1A) from the234

Users' Report

Figure1. A, a cylindrically averaged difference-Patterson
map calculated from the layer-line reflections of X-ray
diffraction patterns from overstretched muscles. B,
orientations of two heads of a myosin crossbrige in therelaxed state. This figure is seen from the Z-band. The zaxis is coincided with the fiber axis. Two heads areshown as a group of red and purple spheres.
References[1] Oshima et al., PF Activity Rep #21, 244 (2004).
[2] Oshima et al., Fibre. Dif. Rev. 13, 25 (2005).
[3] Oshima et al., PF Activity Rep #22, 243 (2005).
[4] Oshima et al., PF Activity Rep #22, 244 (2005).
*waka@bpe.es.osaka-u.ac.jp