Combining temperature perturbations with X-ray crystallography to study dynamic macromolecules: A thorough discussion of experimental methods.

Temperature is an important state variable that governs the behavior of microscopic systems, yet crystallographers rarely exploit temperature changes to study the structure and dynamics of biological macromolecules. In fact, approximately 90% of crystal structures in the Protein Data Bank were determined under cryogenic conditions, because sample cryocooling makes crystals robust to X-ray radiation damage and facilitates data collection. On the other hand, cryocooling can introduce artifacts into macromolecular structures, and can suppress conformational dynamics that are critical for function. Fortunately, recent advances in X-ray detector technology, X-ray sources, and computational data processing algorithms make non-cryogenic X-ray crystallography easier and more broadly applicable than ever before. Without the reliance on cryocooling, high-resolution crystallography can be combined with various temperature perturbations to gain deep insight into the conformational landscapes of macromolecules. This Chapter reviews the historical reasons for the prevalence of cryocooling in macromolecular crystallography, and discusses its potential drawbacks. Next, the Chapter summarizes technological developments and methodologies that facilitate non-cryogenic crystallography experiments. Finally, the chapter discusses the theoretical underpinnings and practical aspects of multi-temperature and temperature-jump crystallography experiments, which are powerful tools for understanding the relationship between the structure, dynamics, and function of proteins and other biological macromolecules.