Abstract
To minimize radiation damage, crystal structures of biological macromolecules are usually determined after rapid cooling to cryogenic temperatures, some 150-200 K below the normal physiological range. The biological relevance of such structures relies on the assumption that flash-cooling is sufficiently fast to kinetically trap the macromolecule and associated solvent in a room-temperature equilibrium state. To test this assumption, we use a two-state model to calculate the structural changes expected during rapid cooling of a typical protein crystal. The analysis indicates that many degrees of freedom in a flash-cooled protein crystal are quenched at temperatures near 200 K, where local conformational and association equilibria may be strongly shifted toward low-enthalpy states. Such cryoartifacts should be most important for strongly solvent-coupled processes, such as hydration of nonpolar cavities and surface regions, conformational switching of solvent-exposed side chains, and weak ligand binding. The dynamic quenching that emerges from the model considered here can also rationalize the glass transition associated with the atomic fluctuations in the protein.