Project Summary Biomotors are protein machines that convert chemical energy to different kinds of mechanical motions essential to cellular functions. The bacteriophage φ29 genome packaging motor is one of the most powerful biomotors reported. It is responsible for packaging the viral genomic double-stranded DNA (dsDNA) into a preformed protein shell (procapsid) using adenosine triphosphate (ATP) as an energy source. Co-PI Guo has focused on addressing basic questions on the mechanisms of assembly and function of the φ29 motor for decades. Recently, the Guo lab proposed a revolving mechanism for the φ29 motor in which DNA revolves rather than rotates during packaging. His lab has also demonstrated the feasibility of engineering and adapting the motor channel for DNA sensing and fingerprinting at the single-molecule level. These findings bring about immense potential of the powerful φ29 motor, but to fully embrace it for future nanotechnological applications will inevitably require a more detailed understanding of how the motor assembles and operates, much of which is only beginning to be elucidated. Major controversies still exist regarding the stoichiometry and architecture of the functional motor complex and the mechanisms by which DNA is translocated. The overarching goal of this proposal is to elucidate the molecular mechanisms of the bacteriophage φ29 dsDNA packaging motor through identification and characterization of the motor complex at a variety of functional states using state-of-the-art cryo-electron microscopy (EM) and molecular dynamics (MD) simulation, and leverage the knowledge gleaned for the design and fabrication of a biologically active ATP-driven procapsid-free nanomotor that provides unprecedented functionality. We propose three specific aims to tackle these challenging problems by integrating advanced cryo- EM (co-PI Mao) and computational (PI Cheng) approaches with well-established biochemical/molecular biology protocols (co-PI Guo) to increase the chances of success and impact of the results. In Aim 1, we will combine cryo-EM and our advanced computational image analysis techniques to identify and characterize a variety of motor intermediates in situ. To achieve high resolutions, we will explore ways to control the dynamics of the motor to obtain structurally more homogeneous specimens. In Aim 2, we will implement and utilize a pipeline of MD simulations to map the various cryo-EM structures obtained in Aim 1 onto the free energy landscapes of the motor complex, and connect them into molecular “movies” to elucidate at the atomic level how stepwise, distributed conformational dynamics in the motor complex are coordinated to drive DNA translocation. Finally in Aim 3, we will leverage the molecular insights into motor assembly and operation gleaned from Aims 1&2 to construct a biologically active procapsid-free φ29 mimetic nanomotor by reconstituting it into a lipid bilayer platform, which will open up enormous opportunities f...