The functions of biological macromolecules mainly depend on their three-dimensional structure, movements, and interactions. Protein structure analysis can provide information on protein classification, protein function, protein interactions, posttranslational modifications, and many other aspects. It fundamentally clarifies the molecular mechanism, and is also an important way to study protein function. At present, there are three main methods to determine the 3D structure of proteins, including X-ray crystallography, nuclear magnetic resonance technology (NMR), and 3D reconstruction technology of cryo-electron microscopy. One of the central goals of synthetic structural biology is to create proteins with novel structures. Therefore, protein structure determination plays a critical role in synthetic biology.