Enhancing Protein Crystallisation with Entropic Mutants and Thermal Shift Assays: A Practical Guide for Structural Biology Students

Protein crystallisation is often the main bottleneck in structural biology. This practical guide introduces a rational approach combining Surface Entropy Reduction (SER) mutagenesis—engineering “entropic” mutants that reduce surface flexibility—with Thermal Shift Assays (TSA) to evaluate and enhance protein stability prior to crystallisation trials. The workflow integrates construct design, buffer optimisation, ligand/partner-protein stabilisation, and TSA-based decision making. This guide is designed for students entering X-ray crystallography, structural biochemistry, and protein engineering, and includes open-access references as well as best practices for experimental setup.