Three-dimensional image reconstruction of rigor crossbridges in insect flight muscle (IFM)

Dorsal longitudinal IFM from the water bug Lethocerus indicus, was rigorized by glycerination. Fiber bundle 958 retained a good rigor x-ray diffraction pattern after a 5 step fixation embedding sequence which included glutaraldehyde-tannic acid fixation, OsO4-UrAc staining, ethanol dehydration and Araldite embedding.The MYAC single filament layer of insect flight muscle contains alternating thick and thin filaments and is found isolated within longitudinal sections about 250Å thick. For our 3-D reconstruction we selected a MYAC region which appeared homogenous (both sides equal) over nearly 3/4 of a half sarcomere. Two tilt series ranging through ±60° of tilt in 10° steps were obtained, with tilt axis parallel to the filament axis in one and perpendicular in the other. Fourier transforms were calculated from 256x256 point arrays.The tilt data were combined using procedures similar to those used for 2-D crystalline arrays except that we obtain both amplitudes and phases from the image data.