CryoEM Single Particle Reconstruction of Dephosphorylated HMM from Smooth Muscle

Abstract

The asymmetric head-head interaction that characterizes the inhibited state of smooth muscle myosin was first discovered in 3-D images of 2-D arrays formed on lipid monolayers at ∼2 nm resolution. The conformation is generally referred to as the “interacting heads motif” or IHM and has since been observed in 3-D images of thick filaments from all species examined from vertebrate striated muscle to the flight muscles of the large waterbug Lethocerus sp. indicating it is a ubiquitous myosin II inhibited conformation. However, current 3-D images reported from filaments are limited to resolutions lower than 1 nm. Here we report on progress toward an atomic resolution structure of the IHM of smooth muscle HMM using single particle methods from dispersions of single particles bound to a lipid monolayer. Data were collected on a Titan Krios equipped with a Volta Phase Plate and a Direct Electron DE-64 detector. Initial reconstructions, starting from a total of 1464 phase plate images from which 588,000 particles were selected using Gautomatch, which is a template matching particle picker, using a template derived from the smooth muscle HMM crystal structure. This structure went to 1.49 nm resolution from a subset of 285300 particles. The free and blocked heads of the IHM are easily distinguishable but the S2 domain is not yet visible. The structure is being refined with addition of new images. Supported by NIH.