Cryo-crystallography

Radiation damage

Just as any organic or living material, proteins are sensitive to X-ray radiation damage. The energy range of X-rays used for diffraction is in the 6 to 15 keV range, which is in fact severely ionizing radiation. The ionizing absorption events create radicals, which rapidly destroy any protein crystal, particularly at dose rates experienced at synchrotrons. An efficient way to suppress radiation damage by slowing down the kinetics of the radical reactions is cryogenic cooling. Rapidly quenching or flash-cooling crystals to liquid nitrogen temperatures, either in cold nitrogen gas streams or directly into liquid nitrogen, will strongly reduce radiation damage. To prevent the formation of crystalline ice during flash-cooling of the crystals, cryoprotectants, present in the mother liqor or added to the mother liquor, are necessary.

Cryo-mounting

 Cryoprotection is effectively accomplished during harvesting, when the crystals are scooped up from the drop in cryo-loops and briefly swept though a cryoprotectant before being dipped into liquid nitrogen. Common cryoprotectants are ethylene glycol (the anti-freeze in automobile radiators), glycerol, higher alcohols, polyethylene glycols (PEGs), or high concentration solutions of sucrose or salts. Once the protein crystals are flash-cooled and stored in pucks in a liquid nitrogen dewar (dry shipper), they can be safely sent to a synchrotron for data collection.

Laser-etched Kapton cryo-loops by MiTeGen are sturdy but still flexible and allow reproducible mounting of crystals down to 10 micron size.

More about sample handling, harvesting and mounting in my book. You can read the first pages 1, 2, 3 of Chapter 8 (Instrumentation and Data Collection) of my book Biomolecular Crystallography or buy the book from Amazon.

Benefits of Cryo-Cooling

Many factors contribute to improvements in data quality during cryo-protection: Obvious benefits are reduced thermal vibrations, enhanced signal-to-noise ratio, reduced conformational disorder, and in many cases, a higher limiting resolution. The most important effect is the suppression of radiation damage, permitting a complete data set to be collected from one single crystal. This in turn eliminates errors from merging and scaling of data sub-sets from multiple crystals or non-isomorphism between MAD data sets. In addition, crystal mounting is vastly simplified over conventional capillary techniques. These improvements combined lead to enhanced contrast and sharper detail in electron density maps, facilitating model building and reducing the total time required for structure determination.

New Crystal harvesting Robots are developed now to facilitate the harvesting of the fragile crystals from their mother liquor.

bullet_2_llow.gif (982 bytes)Robotic crystal harvesting and cryo-cooling movie