EM STUDIES ON MUSCLE

Florida State University, Tallahassee FL

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Abstract

The long term goal of this research project is to understand the molecular mechanism of force production through 3-D visualization of crossbridge states in situ in muscle. The research focuses on the structure of the large waterbug Lethocerus sp. because its filament lattice is the best ordered of all known muscles thereby making it an excellent candidate for 3-D imaging as well as facilitating the trapping of many crossbridges into similar structures. Specimen preparation emphasizes rapid freezing and freeze substitution which traps molecular motions with millisecond time resolution. Crossbridges during isometric and stretch activated contraction are emphasized and complemented by states stabilized with nucleotide analogues. Crossbridges responding to mechanical perturbations such as stretch and release will be trapped by fast freezing and imaged in 3-D. Structures observed by 3-D electron microscopy of sectioned muscle will be correlated with X-ray diffraction of native muscle and mechanical measurements made prior to freezing. Reconstruction work will focus on thin 20-30 nm sections which yield the highest detail on crossbridge structure. We will utilize electron tomography to obtain 3-D images of muscle crossbridges without spatial averaging and use 3-D correspondence analysis to identify groups of similarly structured crossbridges for subsequent averaging to improve the signal to noise ratio. Continued refinements of our unique tomographic method are proposed in order to increase resolution and improve the reconstructions. Atomic models based on the crystal structures of actin and myosin will be built to fit the envelope of the reconstruction and then refined using real space refinement. Finally, we will use these atomic models as a basis for simulating the X-ray diffraction patterns of native muscle.