The structure of insect flight muscle in the presence of AMPPNP.

Glycerinated insect (Lethocerus) flight muscle in the presence of the non-hydrolysable ATP-analogue AMPPNP (1 mM at 4 degrees C) has been prepared for electron microscopy using X-ray diffraction monitoring during fixation and embedding. Superior preservation of the original structure has been achieved through use of a fixative which included tannic acid and excess Mg2+. New features have been recognized in single filament layers (myac and actin) and 15 nm cross sections. As previously shown, some aspects of relaxed structure (14.5 nm shelves along thick filaments) and of rigor (38.7 nm angled bridges along thin filaments) are retained in a modified form. New observations include: (1) In 15 nm cross sections that show single 14.5 nm levels: (a) The flared X structure characteristic of rigor is replaced by a straight-X figure in which the crossbridge density is aligned along the myosin-actin plane, rather than skewed across it as in rigor. (b) In AMPPNP, each crossbridge appears to have a separate origin from the thick filament, rather than bifurcating two from one stem as in the flared X of rigor. The separation of crossbridge origins is also evidenced by the loss of 'ladder rungs' in actin layers. (2) A halving (19.3 nm) of the 38.7 nm axial repeat along actin, rather than a thirding (12.9 nm) as in rigor, indicates redistribution of bridge attachments in cold AMPPNP. (3) In AMPPNP, the 14.5 nm shelves of density around the thick filament shaft are thicker but extend to smaller radius than similar shelves in ATP-relaxed muscle. This is shown by a lack of 14.5 nm periodicity and diffraction in actin layers of AMPPNP, in contrast to ATP-relaxed actin layers, in which the 14.5 nm period is present. Our results suggest that attached crossbridges are modified by AMPPNP and that ordered features of the analogue state are not accounted for solely by detached myosin heads or by a mixture of relaxed and rigor crossbridges. A two-domain model for the crossbridge is proposed. Domain 1 binds to the thin filament, and while bound, maintains a constant stereospecificity to actin at low resolution, independent of the type or presence of nucleotide at the myosin active site. Domain 2 is proximal to the thick filament and can exist in two characteristic states. In the absence of nucleotide (rigor), Domain 2 adopts a variable relationship to the thick filament, to accommodate the actin-bound end of the bridge.(ABSTRACT TRUNCATED AT 400 WORDS)