Computational protocol: Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles, Revealed by Synchrotron X-Ray Fiber Diffraction

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Protocol publication

[…] For atomic fitting studies, an atomic structure of skeletal myosin head (S1) (myosin II S1) in ADP and Pi-bound state (S1.ADP.Pi) (S1, a subfragment 1 part of a myosin molecule) was modeled using the 1072C°-model which was made by modifying the crystal structure of nucleotide-free S1 (PDBID: 2MYS) to yield a better fit to the small-angle x-ray scattering profile of skeletal S1 in MgATP solution , . Then the modified residues of N-dimethyl-lysine of the atomic structure of nucleotide-free S1 were replaced by lysine residues for estimating accurate electrostatic potentials on the surface of S1, but the missing chains in the crystal structure were not restored. The coordinates of main- and side-chain atoms except for C° atoms were modeled by “PULCHRA” (a tool for a full-atom reconstruction and a refinement of reduced protein models) . Energy minimization of an S1.ADP.Pi structure in a vacuum was then done by using the “cosgene Molecular Dynamics (MD) engine of myPresto” . The atomic structure of S1.ADP.Pi was superimposed onto the optimum 68-sphere model using the same parameters as shown in . Since the atomic structure was treated as a rigid body in superimposition, there were many steric clashes of side chains observed in the interface between the heads in the optimum orientation of a myosin crossbridge. Clashes were removed by further energy minimization. A Ramachandran plot was calculated to observe the validity of the atomic model. It is a way of validating an atomic model by seeing whether its C° atom’s dihedral angles of each amino acid are within realistic limits. There is the freedom of rotation about two bonds of amino acids allowing protein to fold in many ways. The rotations about these bonds can be specified by dihedral angles, called φ and ψ. Although there are many combinations of φ and ψ, a Ramachandran diagram visualizes their sterically favored, allowed and other (outlier) values of amino acids in protein as clustered regions on a two-dimensional plot. A structure and a Ramachandran diagram of the energy-minimized S1.ADP.Pi are shown in . This diagram was generated using the CCP4 tool, rampage by Richardson and coworkers . For inspection of the nature of head-head interactions, electrostatic potentials on the surface of each head of the energy-minimized crossbridges were calculated by using the “APBS” tool in PyMOL (The PyMOL Molecular Graphics System, Version 1.5, Schrödinger, LLC.) . […]

Pipeline specifications

Software tools PULCHRA, myPresto, PyMOL
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma