TOMOGRAPHIC 3-DIMENSIONAL RECONSTRUCTION OF INSECT FLIGHT-MUSCLE PARTIALLY RELAXED BY AMPPNP AND ETHYLENE-GLYCOL

Rigor insect flight muscle (IFM) can be relaxed without ATP by increas ing ethylene glycol concentration in the presence of adenosine 5'-[bet a'gamma-imido]triphosphate (AMPPNP). Fibers poised at a critical glyco l concentration retain rigor stiffness but support no sustained tensio n (''glycol-stiff state''). This suggests that many crossbridges are w eakly attached to actin, possibly at the beginning of the power stroke . Unaveraged three-dimensional tomograms of ''glycol-stiff'' sarcomere s show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an av erage 90 degrees axial angle contact actin midway between troponin sub units, which identifies the actin azimuth in each 38.7-nm period, in t he same region as the actin target zone of the 45 degrees angled rigor lead bridges. These 90 degrees ''target zone'' bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges, Another class of glycol-PNP crossbridge binds outs ide the rigor actin target zone. These ''non target zone'' bridges dis play irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into t he tomogram reveals that 90 degrees target zone bridges share with rig or a similar contact interface with actin, while nontarget crossbridge s have variable contact interfaces. This suggests that target zone bri dges interact specifically with actin, while nontarget zone bridges ma y not. Target zone bridges constitute only similar to 25% of the myosi n heads, implying that both specific and nonspecific attachments contr ibute to the high stiffness. The 90 degrees target zone bridges may re present a preforce attachment that produces force by rotation of the m otor domain over actin, possibly independent of the regulatory domain movements.