Progress in rational methods of cryoprotection in macromolecular crystallography

Abstract

Cryogenic cooling of macromolecular crystals is commonly used for X-ray data collection both to reduce crystal damage from radiation and to gather functional information by cryogenically trapping intermediates. However, the cooling process can damage the crystals. Limiting cooling-induced crystal damage often requires cryoprotection strategies, which can involve substantial screening of solution conditions and cooling protocols. Here, recent developments directed towards rational methods for cryoprotection are described. Crystal damage is described in the context of the temperature response of the crystal as a thermodynamic system. As such, the internal and external parts of the crystal typically have different cryoprotection requirements. A key physical parameter, the thermal contraction, of 26 different cryoprotective solutions was measured between 294 and 72 K. The range of contractions was 2-13%, with the more polar cryosolutions contracting less. The potential uses of these results in the development of cryocooling conditions, as well as recent developments in determining minimum cryosolution soaking times, are discussed.

Figure 1

Model for cooling-induced crystal damage. Cooling triggers contraction of the protein, lattice repacking and the consequent contraction and reshaping of the solvent channel (black outline; Juers & Matthews, 2001 ▶). If the internal liquid (light gray) does not contract enough to compensate, the unit cell bursts, much as a copper pipe carrying water can burst if it is cooled below the freezing point of water. Curved black arrows show a hypothetical flow of the liquid. In this example, an idealized perfect crystal is broken into three smaller domains.

Figure 2

Plot of the measured thermal contraction (Δν/ν_RT) versus the calculated polar surface area of the cryosolution. Data are shown for the solutions from Table 1 ▶ at 50%(w/w) cryoprotective agent/water.

Figure 3

Often the crystal-growth buffer (dots) needs to be exchanged for a different solution for successful cooling. The required internal cryoprotective solution (light gray) may be different from the external cryoprotective solution (dark gray).