Improved Imaging, 3D Reconstruction and Homology Modeling of Tarantula Thick Filaments

Abstract

Tarantula muscle is an outstanding model system for elucidating thick filament molecular structure. In past studies we showed by cryo-EM, single particle reconstruction, and atomic fitting with a hybrid myosin head structure, that relaxed tarantula filaments are characterized by intramolecular interaction between heads (J-motif) that is thought to inhibit myosin activity by blocking actin binding in one head and ATPase activity in the other (Woodhead et al., 2005). We have been pursuing improvements in technique to obtain a higher resolution reconstruction. A key problem in reconstructing myosin filaments is intrinsic flexibility of the heads, which reduces the resolution we can achieve. We have used staurosporine to minimize RLC phosphorylation and thus head disordering, and blebbistatin to stabilize the heads. Automated data collection has been used to obtain images on a field emission cryo-electron microscope (Titan Krios). Preprocessing of images has enabled the selection of a data set as homogeneous as possible. Both SPIDER and RELION were used for 3D reconstruction, and yielded a similar resolution of ∼13 A, 2-fold better than our original reconstruction, but presumably still limited by some head flexibility. The J-motif is consistent with the original findings, but the structure of the S2 segment of the myosin tail is much more robust and some details of the paramyosin core are now visible. We have built an homology model of the interacting-heads motif (using the tarantula sequence for both heavy and light chains), that includes all surface loops, and we are using this for molecular dynamics flexible fitting (MDFF). Preliminary results suggest the need for repositioning of several loops from their positions in the crystal structure, with possible implications for filament assembly and stability.