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Cross-Bridge Number, Position, and Angle in Target Zones of Cryofixed Isometrically Active Insect Flight Muscle

Richard T. Tregear ^*, Mary C. Reedy , Yale E. Goldman , Kenneth A. Taylor , Hanspeter Winkler , Clara Franzini-Armstrong , Hiroyuki Sasaki ^¶, Carmen Lucaveche  and Michael K. Reedy 

^* Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom; Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710 USA; Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19194-6083 USA; Institute of Molecular Biophysics, Florida State University, Florida 32306-4380 USA; ^¶ Institute of DNA Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan

Correspondence: Address reprint requests to Dr. Richard Tregear, Structural Studies Division, MRC Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, UK. Tel.: 44-1223-365963; Fax: 44-1223-213556; E-mail: rt1{at}mrc-lmb.cam.ac.uk.

Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.5-nm-spaced shelves along the thick filament to their thin filament attachments in the target zones. The relationship between the probability of cross-bridge formation and axial offset between the shelf and target zone center was well fitted by a Gaussian distribution. One head of each myosin whose origin is close to an actin target zone forms a cross-bridge most of the time. The probability of cross-bridge formation remains high for myosin heads originating within 8 nm axially of the target zone center and is low outside 12 nm. We infer that most target zone cross-bridges are nearly perpendicular to the filaments (60% within 11°). The results suggest that in isometric contraction, most cross-bridges maintain tension near the beginning of their working stroke at angles near perpendicular to the filament axis. Moreover, in the absence of filament sliding, cross-bridges cannot change tilt angle while attached nor reach other target zones while detached, so may cycle repeatedly on and off the same actin target monomer.

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