Macromolecular crystallography provides high-resolution structural information of proteins and protein-ligand complexes, offering essential insights for structure-based drug discovery. Electron diffraction methods, including three-dimensional electron diffraction (3D ED), also known as micro-crystal electron diffraction (MicroED), and serial electron diffraction (SerialED), enable structure determination from nanocrystals and are particularly useful for macromolecules that cannot form large crystals or require long crystallization times. However, the application of electron diffraction to macromolecular structure determination remains challenging, owing to limitations in sample preparation, beam-induced damage, and the efficiency of data processing. To overcome these difficulties, multiple methods and workflows were developed and systematically optimized in this thesis. 

For sample preparation, a rapid mixing protein crystallization (RaMiC) method combined with a surfactant-assisted grid preparation strategy was established. Through this method, cryogenic electron microscopy (cryo-EM) grids containing well-ordered sub-micron-sized crystals suitable for electron diffraction can be reproducibly prepared. To simplify and streamline MicroED data processing, a graphical platform named Automated Real-time and Offline Batch MicroED Data Processing Graphical User Interface (AutoLEI) was developed. It enables high-throughput offline batch processing of large numbers of datasets within minutes and supports online real-time processing. To further reduce electron beam damage, a continuous SerialED (c-SerialED) method was developed, and as a benchmark, the triclinic lysozyme structure was determined at 0.83 Å resolution. From the generated difference map, potential positions for hydrogen atoms were directly identified. 

Based on these developments, a structure-based drug discovery workflow integrating RaMiC, rapid soaking, real-time MicroED ligand screening and high-resolution c-SerialED structure determination was established. To validate the workflow, crystals of the human protein MutT homolog 1 (MTH1) were soaked individually with four ligands, and the method was applied to determine whether they bind to the protein’s active site. With soaking times as short as 3 seconds, binding could be preliminarily evaluated by real-time MicroED within 25-50 minutes. For three ligands that passed pre-screening, c-SerialED data were collected and structures of protein-ligand complexes were solved at 1.7 Å resolution. The electrostatic potential maps clearly revealed the binding modes. 

In summary, this thesis establishes a practical workflow for high-resolution protein and protein-ligand complex structure determination by electron diffraction, highlighting its potential in structure-based drug discovery.