A Tutorial Review on the Methodologies and Theories Utilized to Handle Proteins toward Obtaining Single Protein Crystals

Proteins are functional macromolecules in living organisms including mammals and thus human beings. They are involved in almost all biological processes, either as the main functional domains or as catalysts (enzymes). Owing to their vitality, proteins have attracted extensive research interest from different structural perspectives. Precise determination of the three-dimensional structures of proteins is crucial; otherwise, the structure–function relationships would be ambiguous. Macromolecular crystallographers have been trying to determine protein structures since the 1950s. According to the protein data bank (PDB) on February 13, 2024, 215 684 structures of proteins, nucleic acids, and other biological macromolecules have been determined. Most of these structures (84.6%) were solved using X-ray diffraction (XRD), which requires the growth of protein microcrystals. Only 0.108% and 0.104% of all structures were solved using electron and neutron crystallography methods, which require the growth of nanocrystals and mesoscopic crystals, respectively. Other methodologies such as nuclear magnetic resonance (NMR) (6.5%), electron microscopy (EM) (8.5%), and other biochemical methodologies (