SHOCK FREEZING OF MACROMOLECULAR CRYSTALS AT BEAMLINE FIP

The collection of macromolecular diffraction data has recently benefited from technique and equipment advances which have made low temperature data collection almost like a routine. The ability to freeze protein crystals at liquid nitrogen temperatures normally gives the level of radiation protection necessary to collect complete data sets from single crystals.

To improve the radiation sensitivity of protein samples at such intense X-ray sources as ESRF/FIP has upgraded both its equipment and its capability to offer support to assure that visiting user groups can routinely collect low temperature data while at the synchrotron.

The collection of low temperature data on protein samples hinges around a loop mounting technique that consists of picking crystals up by lifting them in thin loops (fashioned from glass wool, rayon, nylon, silk, chirurgical cotton yarn or any other thin, non-diffracting fibers) by surface tension from soaks of mother liquor containing some cryoprotecting molecule. These molecules were generally freezing point depressing solutes such as sucrose or low molecular weight alcohols (glycol, isopropanol or methylpentanediol (MPD)). In addition to such alcohols, lower molecular weight polyethylene glycols (PEG 400 or 600) have been used successfully. Using such equipment, 360 degrees of continuous rotation data can be collected from a single crystal without worrying about significant shadowing or increases in absorbance or scatter from the loop mounts.

The key to shock freezing crystals is maximizing the efficiency of the initial freezing rate, or the shock. Two methods are currently used to accomplish this. First, crystals can be frozen directly in the cold stream. In this method, a reliable cryostat capable of maintaining a laminar cold stream at -165 degrees to -175 degrees with a good flow rate is essential. Actual freezing involves temporarily deflecting the cold stream with something as simple as a phone card while the crystal is quickly seated in the diffraction position (usually introducing the loop attached to a brass pin into the clamping collar of the goniometric head). Then, the stream deflection is removed and the low temperature gas is used to flash freeze the crystal.

In the second technique, loop mounted crystals are first immersed in some low temperature liquid such as liquid nitrogen, propane, or freon. Liquid propane freezing is the most likely trend of the future as it provides the best thermal capabilities for heat conductance away from the immersed crystal. After immersion, the crystal is seated at the sample position still in the low temperature liquid, and the vessel containing the liquid is quickly removed. In this case, the liquid is used to cause the initial flash and the challenge is smoothly tranferring the frozen crystal to the gaseous cold stream. Both techniques have been used successfully at ESRF/FIP beamline by outside and local user groups.

As has been mentioned, an equally important technique for crystal freezing is the availability of good cryostats. To shock freeze protein crystals we have opted for the Oxford cryostream (600 Series Cryostream Cooler).

At the leading edge of crystal cooling, the cryostream cooler is the Oxford Cryostream nitrogen gas, low temperature attachment for X-ray crystallography. Its unique design means it can be easily fitted to our two types of X-ray systems including image plates, CDD detector. Mounting the Cryostream Coldhead using the Varibeam Coldhead Support Stand means the cold stream can be oriented with precision (X-Y positioner) with the most crowded X-ray enclosures.
The high level of performance from the cryostream cooler means that it encompasses all aspects of low temperature X-ray crystallography. It is perfect for protein and virus crytallographers where it is an essential tool for flash freezing. Cryostream utilises an open flow nitrogen gas stream with an ancillary dry nitrogen shroud to produce a high quality laminar flow system for reliable and ice-free crystal cooling. The cryostream cooler is fed directly from a 55 liters Dewar which is connected to the liquid nitrogen network of ESRF.
A set of 3 probes is coupled with a multi-levels regulator (Air Liquid Inc.) to perform the refilling of the dewar automatically.
The major step to achieving the high temperature stability, low base temperature and low liquid nitrogen consumption is the use of a positive flow gas pump in the principal nitrogen flow circuit.
This means the liquid nitrogen can be sucked from an open Dewar vessel. This ensures that the flow of the nitrogen gas over the crystal is isolated from any disturbances when refilling supply vessel. Furthermore, the cooling ability of the liquid nitrogen is transferred from the Dewar to the Cryostream coldhead at a position very close to where it is needed.
Inside the cryostream coldhead, the liquid nitrogen is evaporated to gas and used to cool one path of a heat exchanger. This gas passes from the coldhead through the gas pump and temperature controller where the flow is regulated and monitored. The gas re-enters the coldhead and is cooled along the second path of the heat exchanger. This cold gas is then heated to the required temperature by a heating element in the cryostream nozzle.
On exiting the nozzle, the cold stream is shrouded by a warm dry gas nitrogen to prevent icing at the crystal. The cryostream cooler is controlled and monitored by a purpose built microprocessor based system manager and three term (PID) temperature controller. The controller is designated to allow the easy input of different programs through a simple set of keys in response to on-screen prompts. By using a set of in-built modes it is possible to program in sophisticated combinations of ramps and plateaus to control the temperature of the gas stream in any desired way. A built-in RS232 interface enables the controller to be interrogated or programmed remotely.