Cryoem Structure Determination of Actin Decorated with Smooth Muscle Heavy Meromyosin
Social Science Research Network(2022)
Abstract
The 3-D structure of F-actin in complex with recombinant smooth muscle heavy meromyosin in the presence of Mg·ADP has been determined by cryoelectron microscopy. Specimens were prepared using a NI-NTA lipid monolayer combined with a C-terminal His tag on the myosin to prevent diffusion to the air-water interface. Using iterative helical real space reconstruction, we aligned decorated actin segments offset successively along the F-actin genetic helix in order to align the actin subunits and myosin motor domains as accurately as possible. Identification of two-headed, heavy meromyosin actin contacts was obtained using masked 3-D classification of this aligned data set. Despite a relatively low yield of 2-headed single filament class averages, the resolution of the lever arm portion of the myosin head was superior to that typically obtained from actin decorated with S1 alone. The paired myosin lever arms differ slightly in structure at the converter domain and essential light chain. Significantly, the lever arms differ more in structure at the location of the regulatory light chain indicating a point of flexibility in the myosin heavy chain between the essential and regulatory light chains. Approximately one third of the twenty members of the myosin superfamily of molecular motors contain a pair of heavy chains and may also be amenable this type of structural analysis.
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Key words
actin decorated,muscle
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