3-DIMENSIONAL STRUCTURE OF NUCLEOTIDE-BEARING CROSSBRIDGES IN-SITU - OBLIQUE SECTION RECONSTRUCTION OF INSECT FLIGHT-MUSCLE IN AMPPNP AT 23-DEGREES-C

We have explored the three-dimensional structure of myosin crossbridge s in situ in order to define the structural changes that occur when nu cleotide binds to the myosin motor. When AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by similar to 2.0 nm and tension is reduced by similar to 70% without a reduction in stiffness, suggesting partial reversal of the power stroke. We have obtained ave raged oblique section three-dimensional reconstructions of mechanicall y monitored insect flight muscle in AMPPNP that permit simultaneous ex amination of all myosin crossbridges within the unit cell and direct c omparison of calculated transforms with X-ray diagrams of the native f ibers. Transforms calculated from the oblique section reconstruction o f AMPPNP insect night muscle at 23 degrees C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms i n 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 th e position of the rigor lead bridge. The portion of the crossbridge cl ose to the thick filament appears broader than in rigor, and shows a s mall 0.5 to 1.0 nm M-ward shift of the regulatory domain region of myo sin. in transverse view, AMPPNP ''lead'' crossbridges are less azimuth ally bent than rigor. Fitting the atomic model of actomyosin subfragme nt 1 to the averaged crossbridges shows that the detectable difference s between rigor bridges and between rigor and AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that r ear bridges are more strained than lead crossbridges. The apparent abs ence of rear bridges in AMPPNP in averaged reconstructions indicates d etachment of a number of force-bearing bridges, which conflicts with t he maintained stiffness of the fibers used for the reconstruction. Thi s conflict may be explained if rigor rear bridges become distributed i rregularly over more actin sites in AMPPNP, so that their average dens ity is too low to appear in the averaged reconstructions. The reconstr uctions indicate that in insect flight muscle the response of in situ rigor crossbridges to AMPPNP binding is not uniform. Lead bridges pers ist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached m yosin heads within the myofibrillar lattice are in the appropriate dir ection and of the appropriate magnitude needed to explain the sarcomer e lengthening. This could be a direct response to nucleotide binding, a passive response to rear bridge detachment, or a combination of both . (C) 1996 Academic Press Limited