Visualization of macromolecular structures is essential for understanding the mechanisms of biological functions because they are all determined by the structure and dynamics of macromolecular complexes. Electron cryomicroscopy (cryoEM) and image analysis has become a powerful tool for structural studies because of recent technical developments in microscope optics, cryostage control, image detection and the methods of sample preparation. In particular, the recent development of CMOS-based direct electron detectors with high sensitivity, high resolution and high frame rate has revolutionized the field of structural biology by making near-atomic resolution structural analysis possible from small amounts of solution samples. However, for some biological systems, it is still difficult to reach high resolution due to somewhat flexible nature of the structure, and a complementary use of cryoEM with X-ray crystallography is essential and useful to gain mechanistic understanding of the biological functions and mechanisms. We will describe our strategy for the structural analyses of actin filament and actomyosin rigor complex and the biological insights we gained from these structures.
Keywords: 3D reconstruction; Actomyosin motor; Biased Brownian motion; Electron cryomicroscopy; F-actin assembly; Hybrid method for structural analysis; Image analysis; Skeletal muscle contraction; Treadmill; X-ray crystallography.