We have explored the three-dimensional structure of
myosin crossbridges in situ in order to define the structural changes that occur when
nucleotide binds to the
myosin motor. When
AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by approximately 2.0 nm and tension is reduced by approximately 70% without a reduction in stiffness, suggesting partial reversal of the power
stroke. We have obtained averaged oblique section three-dimensional reconstructions of mechanically monitored insect flight muscle in
AMPPNP that permit simultaneous examination of all
myosin crossbridges within the unit cell and direct comparison of calculated transforms with X-ray diagrams of the native fibers. Transforms calculated from the oblique section reconstruction of
AMPPNP insect flight muscle at 23 degrees C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms in the reconstruction reflect the native structure. In contrast to the rigor lead and rear crossbridges in the double chevrons, the averaged reconstruction of
AMPPNP fibers show only one crossbridge class, in the position of the rigor lead bridge. The portion of the crossbridge close to the thick filament appears broader than in rigor, and shows a small 0.5 to 1.0 nm M-ward shift of the regulatory domain region of
myosin. In transverse view,
AMPPNP "lead" crossbridges are less azimuthally bent than rigor. Fitting the atomic model of
actomyosin subfragment 1 to the averaged crossbridges shows that the detectable differences between rigor bridges and between rigor and
AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that rear bridges are more strained than lead crossbridges. The apparent absence of rear bridges in
AMPPNP in averaged reconstructions indicates detachment of a number of force-bearing bridges, which conflicts with the maintained stiffness of the fibers used for the reconstruction. This conflict may be explained if rigor rear bridges become distributed irregularly over more actin sites in
AMPPNP, so that their average density is too low to appear in the averaged reconstructions. The reconstructions indicate that in insect flight muscle the response of in situ rigor crossbridges to
AMPPNP binding is not uniform. Lead bridges persist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached
myosin heads within the myofibrillar lattice are in the appropriate direction and of the appropriate magnitude needed to explain the sarcomere lengthening. This could be a direct response to
nucleotide binding, a passive response to rear bridge detachment, or a combination of both.