3D Structure of Myosin Crossbridges in Insect Flight Muscle

Abstract

Insect flight muscle (IFM) provides a model system that allows direct viewing of individual myosin head structures in situ that give rise to the average structures reported by X-ray patterns and by the mechanical behavior of the fibers. Coordinating X-ray diffraction, physiological monitoring and fast freezing with EM tomography, correspondence class averaging and atomic model building in IFM is providing 3D imaging of different myosin conformations in situ in relaxed, active and rigor states. Rigor has yielded the most detailed 3D structure, showing actin, myosin S2 and a distribution of variously flexed myosin lever arm in class averages. EM tomograms of fast frozen/freeze substituted isometric and stretch-activated contractions show that crossbridges in active contraction bind to actin target zones by only one head, in contrast to the most prominent class of rigor crossbridges that attach with both myosin heads to actin. In contrast to a ∼5nm lever arm swing inferred during rigor induction, active myosin heads display a wide range of crossbridge angles, consistent with a power stroke greater than 10nm, that proceeds from a prestroke “up” configuration “down” to a rigor angle. However, measurements of isometrically active IFM crossbridges to determine their position, angle and frequency of attachment to actin indicate that the majority of crossbridges in isometric contraction are angled close to perpendicular to the filament axis (60% within 11°), results that are consistent with X-ray studies of vertebrate isometric contraction. X-ray modeling of ATP relaxed Lethocerus IFM shows that a myosin head conformation similar to “prestroke” crystal structures, is arrayed in the 14.5nm periodic “shelves” along thick filaments such that only one head of each molecule is well-positioned, as if poised to bind to actin upon activation, while the other head curves around the thick filament shaft.