Abstract
We have obtained a 4.3A resolution 3-D image of the relaxed thick filament from Lethocerus indicus using IHRSR. The image resolution is variable being highest for the region where the myosin rods are packed and lowest for the blocked head within the interacting heads motif (IHM). Although the resolution is low for de novo atomic model building, by using coiled-coil constraints within the model building software Rosetta, we obtained a complete atomic model for the Lethocerus flight muscle myosin rod, its least understood domain. For the rod, we started with a homology model of the Skip 1 crystal structure (PDB 4XA1) and built outward to the N- and C-termini. The myosin rods assemble into “curved molecular crystalline layers” (ribbons) and not as helical subfilaments. The number of plausible salt bridges between coiled-coils within ribbons is 30% greater than the number between ribbons. For the IHM, we used flexible fitting starting with an homology model of the Lethocerus myosin sequence and the scallop transition state structure, PDB 1DFL. Notable features of the IHM atomic model include the following: (1) Hydrophobic content in the free head RLC interaction with the myosin rods. This is the best-ordered region of the IHM. (2) At the free head S1/S2 junction, 11 residues of the heavy chain α-helix are unwound. Density connecting the displaced parts of the heavy chain α-helix is clearly resolved. (3) The heavy chain α-helices bound by the free head ELC and RLC could not be connected without unwinding 10 residues of the heavy chain α-helix. The blocked head density is the lowest resolution region of the reconstruction and could be fit without the heavy chain helical alterations seen in the free head. Supported by NIH and AHA.
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