CRYOCRYSTALLOGRAPHY OF BIOLOGICAL MACROMOLECULES

P. Řezáčová¹

¹Department of Gene Manipulation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague 6, Czech Republic

Introduction

Crystals of biological macromolecules at near to room temperature are sensitive to X‑rays and frequently suffer from radiation damage, especially when X-ray experiments are carried out on highly intense synchrotron beamlines. Performing such experiments at cryogenic temperatures greatly reduce, or eliminate radiation damage and thus produce higher quality diffraction data. Since about 1985, the cryocrystallography methods have become widely used and well established technique. A brief discussion of the most important experimental aspects and advantages of data collection at low temperature is given. Reviews on cryogenic techniques in macromolecular crystallography can be found in: [1, 2, 3].

Radiation damage

Radiation damage of biocrystals appears to be related to the formation of free radicals. Although the photochemical processes producing free radicals (primary radiation damage) are localized event; subsequent chemical reactions can be induced at relatively remote sites due to the propagation of free radicals in the solvent regions of a protein crystal via diffusion (secondary radiation damage) [4]. Damage is spread and leads to crystal decay, typically accompanied by changes in reflection profiles and cell dimensions.

It was noted as early as 1970 [5] that performing diffraction experiments on protein crystals cooled to near liquid-N₂ temperature leads to significant reduction in radiation damage. This effect is due to facts that by lowering the temperature diffusional processes and therefore propagation of higly reactive free radicals within the crystals is slowed down.

Experimental setup

A schematic of a typical experimental setup of a diffraction experiment at cryogenics temperature is shown in Figure 1. Instead of being sealed in capillary, the crystal is mounted in a thin nylon fiber loop and rapidly cooled, either in a cold gas stream, or by immersion in a cryogen such as liquid nitrogen. The temperature of the crystal is maintained by a stream of nitrogen gas during the diffraction measurement. To avoid the formation of ice around the crystal, the nitrogen stream is shielded agains humidity by a coaxial stream of warm dry air.

Cryoprotection of crystals

When a crystal of biological macromolecule is cooled to cryogenic temperature the main difficulty is to avoid the crystallization of any water present in the system, whether internal or external to the crystal. Therefore a cooling procedure has to be chosen that leads to a glass-like amorphous phase of the solvent [6]. In principle there are three options: (1) cooling on the timescale too fast for ice formation to occur [7], (2) cooling at high pressure by which the formation of common hexagonal form of ice is circumvented [8], (3) modifying the physicochemical properties of the solvent by addition of cryoprotectants in a way that vitrified state can be reached at moderate cooling rates. The latter method is currently the most widely used. A list of cryoprotectants used successfully with macromolecular crystals is shown in Table 1 [9]. Glycerol appears to be a widely applicable cryoprotectant and is frequently chosen for initial trials [10]. Typically cryoprotectants are included in the established harvesting solution at concentration range 50%. Methods for introducing the cryoprotectants are: (1) serial transfer into increasing concentrations of cryoprotectant, (2) dialysis, (3) growth in cryoprotectant, (4) brief transfer before flash cooling, (4) direct transfer into full strenght cryoprotectant. It is rare that crystal can be transferred without damage directly to a solution containing full-strenght cryosolvent. Usually, the cryoprotectant must be introduced slowly to reduce stress on the crystal lattice. Finding suitable cryoprotection conditions is a trial-and-error process. Two problems must be overcome: the cryoprotectant must be introduced without significant damage to the crystal, and damage during the flash cooling must be minimized.

Crystal mounting and data collection

To facilitate rapid heat transfer the crystal must be in immediate contact with the cooling medium and therefore capillaries cannot be used for mounting. Depending on the mechanical properties of the crystal, glass fibres, glass spatulas or, currently most widely fibre loops are used [11]. Using the nylon fibre loop, protein crystal is picked up by swiftly moving the loop alongside the crystal from the crystallization mother liquor. The crystal is held within the film by surface tension and after equilibration in cryoprotective buffer must be cooled to cryogenic conditions as soon as possible. A simple and often effective approach is to flash cool the crystal in a goniostat nitrogen stream right on the X-ray camera. This technique has the added advantage in leaving the crystal in position for immediate analysis and data collection. An alternative method, rapidly plunging the crystal into a liquid cryogen, also offers several advantages. It reduces the time between mounting the crystal and flash cooling, it produces higher cooling rate and results in more even cooling of both sides of loop-mounted sample. Crystal flash cooled in a liquid nitrogen must be placed for data collection in the cold gas stream a goniostat without any substantial warming.

Storage and transport of crystals

Once a crystal has been successfully cooled to cryogenic temperature it can be in principle stored for indefinite time. This allows to cool and characterize crystals on a conventional source in the home laboratory and then store them until synchrotron time becomes available. Dewars that can be used for transport, including shipment by airplane, are available.

Conclusion and perspectives

Cryogenic methods provide great advantages in macromolecular crystallography especially when synchrotrone radiation is used for diffraction data collection. Apart from eliminating the problem with radiation damage and enabling the storage and safe transport of frozen crystals, there are number of additional benefits. In general higher quality data can be achieved and in many cases all data can be collected from a single crystal. Cryogenic data collection has allowed efficient phasing using multiwavelength methods. Additionally, a crystal at cryotemperature is rigidly attached to its mount, so that slippage during the measurement is impossible.

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8. U. F. Thomanek, F. Parak, R. L. Mossbauer, H. Formanek, P. Schwager, W. Hope, Acta Cryst. A29 (1973) 263

9. D. V. Rodgers, in International Tables for Crystallography Vol. F (2001) 202

10. E. F. Garman, E. P. Mitchell, J. Appl. Cryst. 29 (1996) 584

11. T.-Y. Teng, J. Appl. Cryst. 31 (1998) 252

Figure 1. Schematic view of a typical experimental setup of diffraction experiment at cryogenics temperature

Table 1.

List of cryoprotectans used successfully for flash cooling of biological crystals
Erythriol	2-Metyl-2, 4-pentanediol (MPD)
Ethanol	Polyethylen glycol 400
Ethylen glycol	Polyethylen glycol 1000 – 10 000
Glucose	Propylene glycol
Glycerol	Sucrose
Methanol	Xylitol