Facilitating protein crystal cryoprotection in thick-walled plastic capillaries by high-pressure cryocooling

Abstract

Many steps in the X-ray crystallographic solution of protein structures have been automated. However, the harvesting and cryocooling of crystals still rely primarily on manual handling, frequently with consequent mechanical damage. An attractive alternative is to grow crystals directly inside robust plastic capillaries that may be cryocooled and mounted on the beamline goniometer. In this case, it is still desirable to devise a way to cryoprotect the crystals, which is difficult owing to the poor thermal conductivity of thick plastic capillary walls and the large thermal mass of the capillary and internal mother liquor. A method is described to circumvent these difficulties. It is shown that high-pressure cryocooling substantially reduced the minimal concentrations of cryoprotectants required to cryocool water inside capillaries without formation of ice crystals. The minimal concentrations of PEG 200, PEG 400 and glycerol necessary for complete vitrification under pressure cryocooling were determined.

Figure 1

Diffraction images and diffraction intensity profiles of successful (a), (b) and unsuccessful (c), (d) cryocooling trials. Samples having a trace of crystalline ice features, indicated by the arrows in (c) and (d), are regarded as unsuccessful trials. The inset in (c) is shown at enhanced contrast to show more clearly a sharp but weak crystalline ice line. Note that the scattering features of polycarbonate walls of the capillaries (innermost diffuse ring) seen in (a) and (c) were subtracted (see text for detail) and not shown in (b) and (d). The primary broad peaks in (b) and (d) are from the amorphous water phase.

Figure 2

Success rate of vitrification versus concentration for high-pressure cryocooled PEG 200, PEG 400 and glycerol solutions. A ∼70% success rate is achieved by 8%(v/v) glycerol and 10%(v/v) PEG 400. The curves are low-order polynomial fits simply to guide the eye.

Figure 3

Diffuse peak position of vitreous ice as a function of concentration. The curves are low-order polynomial fits simply to guide the eye, and the data point for pure water (i.e. 0%v/v) was obtained from Tulk et al. (2002 ▶).

Figure 4

X-ray diffraction pattern of a high-pressure cryocooled RNase A crystal grown in the presence of 10%(v/v) glycerol inside a polycarbonate capillary. The crystal diffracted beyond 2 Å and the mosaicity is 0.4°. Note the absence of sharp crystalline ice lines, which indicates successful cryocooling.