International
Tables for
Crystallography
Volume C
Mathematical, physical and chemical tables
Edited by E. Prince

International Tables for Crystallography (2006). Vol. C. ch. 3.4, pp. 166-167

Section 3.4.1.5.3. Crystal mounting and cooling

P. F. Lindleya

a ESRF, Avenue des Martyrs, BP 220, F-38043 Grenoble CEDEX, France

3.4.1.5.3. Crystal mounting and cooling

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Experience indicates that small crystals are better for cryogenic purposes, presumably because the rate of diffusion of small molecules and the rate of heat loss during rapid freezing is significantly faster than for large crystals. In most cases, there is an increase in the mosaicity (typically by a factor of 2–3), and in large specimens the increase may render the crystals useless for data collection. Successful freezing is often indicated by the crystal remaining transparent. Opacity usually indicates considerable breakdown in the crystallinity. Three commonly used methods for mounting crystals of biological macromolecules for cryogenic measurements are detailed below.

  • (i) Coating methods. Useful accounts of this method are given by Dewan & Tilton (1987[link]) and Hope (1988[link]). The crystal is first transferred to a hydrocarbon environment, mounted on a glass fibre attached to a brass pin on a goniometer head, and then fast cooled by introduction into a nitrogen-gas stream. The crystal adheres to the fibre by surface-tension effects, and the hydrocarbon also prevents loss of solvent during transfer into the gas stream. Paratone-N (Exxon) mixed with mineral oil (25–50% mineral oil) has a suitable viscosity, and excess oil should be removed by draining. This method has been successfully used for a number of biological macromolecules including crambin (Teeter, Roe & Heo, 1993[link]) and the bovine eye lens protein, γB-crystallin (Lindley et al., 1993[link]). In the case of γB-crystallin, it was found that large crystals, 0.5 × 0.5 × 1.0 mm, often became opaque after freezing, indicating gross damage to the crystallinity, or showed appreciable mosaic spread in the subsequent diffraction patterns, rendering them useless for data collection. Smaller crystals, 0.2 × 0.2 × 0.8 mm, gave good diffraction patterns with an increase in the mosaic spread of only a factor of about two, compared with room-temperature measurements, presumably because of smaller angular and size distributions of the mosaic blocks. For γB-crystallin, the effective resolution was extended from 1.5 Å to at least 1.2 Å. A coating and flash-freezing method has been employed to obtain data from physically fragile and very radiation sensitive crystals of 50S ribosomal particles (Hope et al., 1989[link]). The crystals were transferred to an inert hydrocarbon environment, or to solutions similar to the crystallization medium but with higher viscosities, and flash frozen on a thin glass spatula by immersion in liquid propane. They were then transferred to a cold-nitrogen-gas stream for data measurement. The immersion in a slurry of propane near its melting point gives good wetting of the crystal surface and a heat transfer rate appreciably faster than direct introduction into a cold-gas stream. Transfer from the propane to the gas stream has to be achieved rapidly to avoid ice formation on the surface of the protein owing to condensation of moist air.

  • (ii) Loop techniques. Loops (Teng, 1990[link]; Gamblin & Rogers, 1993[link]), made from fine wire, glass, and a range of thin fibres, can provide very useful mounts for cryocrystallography. Typically, the loops are folded and the two ends glued inside a glass capillary mounted on a goniometer head. Rayon and hair fibres give relatively low backgrounds in diffraction patterns and can readily be made into loops with diameters from 200 to 800 µm. Larger-diameter loops tend to fold over, and glass fibres are more appropriate. Wire loops have a distinct disadvantage in that a plane of diffraction data in which the X-rays are blocked by the wire loop is inaccessible. The diameter is chosen so that the crystal just fits inside the loop and is held in place by surface tension with a thin film of the crystallization/cryoprotectant buffer. The loop with the crystal can then be flash frozen by immersing in liquid propane or fast frozen by direct introduction into a cold-gas stream. Hope (1990[link]) describes a device that can rapidly transfer crystals mounted in loops from a liquid-propane bath to the cooled-gas stream. Indeed, once crystals have been frozen in loops they can be transferred to liquid-nitrogen containers and kept almost indefinitely. A typical application of the loop technique is provided by the crystal structure determination of an extracellular fragment of the rat CD4 receptor (Lange et al., 1994[link]).

  • (iii) Liquid-helium cryostat: neutron diffraction. Slow freezing using a liquid-helium cryostat (Archer & Lehmann, 1986[link]), over a period of hours, has been successfully used with crystals of the coenzyme of vitamin B12 to 15 K (Bouquiere, Finney, Lehmann, Lindley & Savage, 1993[link]), where the solvent content is relatively low, 16–17 water molecules per asymmetric unit. Whether biological macromolecular crystals can be annealed to low temperatures with progressive sets of cooling, heating and cooling stages is not well researched.

References

First citation Archer, J. M. & Lehmann, M. S. (1986). A simple adjustable mount for a two-stage cryorefrigerator on an Eulerian cradle. J. Appl. Cryst. 19, 456–458.Google Scholar
First citation Bouquiere, J. P., Finney, J. L., Lehmann, M. S., Lindley, P. F. & Savage, H. F. J. (1993). High-resolution neutron study of vitamin B12 coenzyme at 15 K: structure analysis and comparison with the structure at 279 K. Acta Cryst. B49, 79–89.Google Scholar
First citation Dewan, J. C. & Tilton, R. F. (1987). Greatly reduced radiation damage in ribonuclease crystals mounted on glass fibres. J. Appl. Cryst. 20, 130–132.Google Scholar
First citation Gamblin, S. J. & Rogers, D. W. (1993). Some practical details of data collection at 100 K. In Data collection and processing. Proceedings of the CCP4 Study Weekend, edited by L. Sawyer, N. Isaacs & S. Bailey. Report DL/SCI/R34. SERC Daresbury Laboratory, Cheshire WA4 4AD, England. Google Scholar
First citation Hope, H. (1988). Cryocrystallography of biological macromolecules: a generally applicable method. Acta Cryst. B44, 22–26.Google Scholar
First citation Hope, H. (1990). Crystallography of biological macromolecules at ultra-low temperatures. Ann. Rev. Biophys. Biophys. Chem. 19, 107–126.Google Scholar
First citation Hope, H., Frolow, F., van Böhlen, K., Makowski, I., Kratky, C., Halfon, Y., Danz, H., Bartels, K. S., Wittmann, H. G. & Yonath, A. (1989). Crystallography of ribosomal particles. Acta Cryst. B45, 190–199.Google Scholar
First citation Lange, G., Lewis, S. J., Murshudov, G. N., Dodson, G. G., Moody, P. C. E., Turkenburg, J. P., Barclay, A. N. & Brady, R. L. (1994). Crystal structure of an extracellular fragment of the rat CD4 receptor containing domains 3 and 4. Structure, 2, 469–481. Google Scholar
First citation Lindley, P., Najmudin, S., Bateman, O., Slingsby, S., Myles, D., Kumaraswamy, S. & Glover, I. (1993). Structure of bovine γB-crystallin at 150 K. J. Chem. Soc. Faraday Trans. 89, 2677–2682.Google Scholar
First citation Teeter, M. M., Roe, S. M. & Heo, N. H. (1993). Atomic resolution (0.83 Å) crystal structure of the hydrophobic protein crambin at 130 K. J. Mol. Biol. 230, 292–311.Google Scholar
First citation Teng, T. Y. (1990). Mounting of crystals for macromolecular crystallography in a free-standing thin film. J. Appl. Cryst. 23, 387–391. Google Scholar








































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