First, a model of actin filament we built on our 3.6 Å resolution map (PDB ID: 5JLF)23 (link) was fitted into the 3D map of the thin filament as a rigid body. For modeling Tm structure, a homology model of Tm was constructed with the full length Tm crystal structure determined at 7 Å resolution (PDB: 1C1G)12 (link) as a template and fitted it into the 3D map. The Tn core domain was fitted by one of the crystal structures of human cardiac Tn in the Ca2+ bound state (PDB: 4Y99)13 (link). Since the resolution of the density of TnCN was not high enough to perform flexible fitting, we divided the Tn core into three domains, TnCN, TnCC, and IT arm, and treated them as rigid bodies. Since the Tm-Tm junction was clearly resolved in the 3D map, it was possible to place the N- and C-termini of four TM chains into the junction to build a model of Tm for its entire length. We then subtracted the model densities of actin filament and Tm from the 3D map of the thin filament to produce a difference map, which revealed the densities for the remaining chains of an N-terminal region of TnT (TnTN) and the C-terminal region of TnI (TnIC). For modeling TnTN, an α-helix model of TnTN residues 87–150 was built by MODELLER36 , and the interactions between the C-terminal end of rabbit skeletal Tm and a short fragment of chicken skeletal TnT revealed in the crystal structure (PDB: 2Z5H)21 (link) was used to build a model of Tm–TnTN complex. For TnIC, the difference map showed an elongated density along actin filament and Tm coiled coil above the Tn core only in the Ca2+ free state. We used the long C-terminal α-helix visualized in one of the crystal structure of human cardiac Tn (PDB: 1J1E)13 (link) to fit into the difference map and modeled the remaining C-terminal region as an extended chain. We used RosettaCM19 (link) for all the modeling and refinement to remove clashes between actin, Tm and Tn and keeping their stereochemistry and used UCSF Chimera for the preparation of all the figures37 (link).
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