Optimization of cryogenic cooling of protein crystals

Structure determination of proteins can be achieved by using X-ray crystallographic techniques. During exposure of protein crystals to ionizing X-rays free radicals are formed, which can alter or damage structure of the protein. Protein crystals contain a significant fraction of solvent which is primarily water allowing rapid diffusion of free radicals when data are collected at room temperature. Radiation damage to protein crystals is greatly reduced with X-ray diffraction data collection at cryogenic temperatures, where the crystal and solvent form a solid matrix. When using low temperatures for X-ray data collection solvent should be vitrified (no crystalline ice formation). Ice disrupts the crystal lattice resulting in poor quality diffraction data. For vitrification of the solvent, protein crystals are "flash cooled" to cryogenic temperatures with liquid or gaseous cryogens such as nitrogen or helium. Cryoprotectants such as glycerol are added to raise the glass transition temperature and lower the ice nucleation temperature of the solvent in and around the crystal to lower cooling rates needed to vitrify samples.; Finding the optimum cryoprotectant concentration for successful flash cooling is largely a trial and error exercise. Valuable protein crystals are often lost in the flash cooling trials. Several factors such as sample size, composition and cryogen type were assessed. A theoretical basis for prediction of cryoprotectant concentration for successful flash cooling was developed.; Investigation of crystal soak protocols in the glycerol solutions showed that crystals should be soaked for sufficient time to allow diffusion of penetrating cryoprotectants into the crystal channels to provide adequate cryoprotection. The minimum soak time needed can be estimated through a calculation of 'penetration' time.; Cryogenic diffraction data collection is normally done at a temperature of ∼ 100 K using a flowing nitrogen gas stream. Use of cryogenic helium, which has a lower (4 K) boiling point than nitrogen (77 K), offers an alternative. Radiation damage to crystals exposed to a high intensity synchrotron beamline using helium as cryogen (cooling agent) was compared with conventional cooling using nitrogen. It was found that use of cryogenic helium results in significant improvement in crystal lifetime and reduction of radiation damage.