Single-particle cryo-electron microscopy (cryo-EM) analysis plays an essential role in the structural determination of protein complexes that are not suitable for crystallization. Technological and methodological advances have promoted the “resolution revolution”, leading to a boom in structures determined by cryo-EM. In this dissertation, I describe the molecular mechanisms of four proteins by determining the structures of them and their complexes at a near-atomic resolution (3–5 Å). These studies demonstrate the essential roles of single-particle cryo-EM analysis in structural biology and protein sciences. I also describe the future development of cryo-EM and structural biology, with advances in automated blotless sample vitrification, real-time data processing, and more accurate de novo protein structure prediction.