Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction.
The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.
Liu
Jun
J
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA.
Wendt
Thomas
T
Taylor
Dianne
D
Taylor
Kenneth
K
D000818:Animals; D002645:Chickens; D020285:Cryoelectron Microscopy / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000379:methods; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D009130:Muscle, Smooth / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation
D008055:Lipids; D009218:Myosins