A simple and rapid preparation of smooth muscle myosin 2 for the electron microscopic analysis.

There has been an increase in the demand for purified protein as a result of recent developments in the structural biology of myosin 2. Although promising, current practices in myosin purification are usually time-consuming and cumbersome. The reported increased actin to myosin ratio in smooth muscles adds to the complexity of the purification process. Present study outlines a streamlined approach to isolate smooth muscle myosin 2 molecules from actomyosin suspension of chicken gizzard tissues. The procedure entails treating actomyosin for a brief period with actin-binding peptide phalloidin, followed by co-sedimentation and short column size exclusion chromatography. Typical myosin molecule with heavy and light chains and approximately 95% purity was examined using gel electrophoresis. Negative staining electron microscopy and image processing showed intact 10S myosin 2 molecules, proving that phalloidin is effective at eliminating majority of actin in the form of F-actin without dramatic alteration in the structure of myosin. The entire purification discussed here can be completed in a few hours, and further analysis can be done the same day. Thus, by offering quick and fresh supplies of native myosin molecules suited for structural research, specially cryo-electron microscopy, this innovative approach can be adapted to get around the drawbacks of time-intensive myosin purifying processes.

Review on the structural understanding of the 10S myosin II in the era of Cryo-electron microscopy.

The compact smooth muscle 10S myosin II is a type of a monomer with folded tail and the heads bending back to interact with each other. This inactivated form is associated with regulatory and enzymatic activities affecting myosin processivity with actin filaments as well as ATPase activity. Phosphorylation by RLC can however, shuttle myosin from the inhibited 10S state to an activated 6S state, dictating the equilibrium. Multiple studies contributed by TEM have provided insights in the structural understanding of the 10S form. However, it is only recently that the true potential of Cryo-EM in deciphering the intramolecular interactions of 10S myosin state has been realized. This has led to an influx of new revelations on the 10S inactivation, unfolding mechanism and association in various diseases. This study reviews the gradual development in the structural interpretation of 10S species from TEM to Cryo-EM era. Furthermore, we discuss the utility of Cryo-EM in future myosin 10S studies and its contribution to human health.

Liquid electron microscopy: then, now and future.

Contemporary microscopic imaging at near-atomic resolution of diverse embodiments in liquid environment has gained keen interest. In particular, Electron Microscopy (EM) can provide comprehensive framework on the structural and functional characterization of samples in liquid phase. In the past few decades, liquid based electron microscopic modalities have developed tremendously to provide insights into various backgrounds like biological, chemical, nanoparticle and material researches. It serves to be a promising analytical tool in deciphering unique insights from solvated systems. Here, the basics of liquid electron microscopy with few examples of its applications are summarized in brief. The technical developments made so far and its preference over other approaches is shortly presented. Finally, the experimental limitations and an outlook on the future technical advancement for liquid EM have been discussed.