Electron Tomography of Swollen Rigor Fibers of Insect Flight Muscle Reveals the S2 Fragment of Myosin

Abstract

We have used electron tomography of rigorized fibers of insect flight muscle swollen by immersion in dilute buffer (†1mM MOPS, pH 6.8), to reveal the 3-D arrangement of the myosin S2 fragment for the first time. S2 is an α-helical coiled-coil structure that connects the myosin head to the thick filament backbone. It is usually invisible in electron micrographs because of its close proximity to the backbone. The increase in inter-thick filament spacing from 45 nm to 55 nm pulls the S2 away from the filament backbone making it easier to identify. A total of 4 tomograms have been calculated by our standard method which involves collecting images using non-uniformly spaced tilt angles, cross correlation alignment, area matching and Whittaker-Shannon interpolation of the 3-D transform. A projection of one of these tomograms is shown in Figure 1. in raw tomograms S2 appears as a rod-like structure that originates at the thick filament surface to connect to both lead and rear crossbridges of rigor. S2 segments are particularly well resolved at the ends of the thick filament (Fig. 2), where in some cases it appears that the myosin subfilaments are separated from the backbone. Both the azimuthal direction and axial angle of S2 are variable.