Tables for
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 10.1, pp. 242-244   | 1 | 2 |

Section 10.1.2. Beneficial effects of low temperature

H. Hopea* and S. Parkinb

aDepartment of Chemistry, University of California, Davis, One Shields Ave, Davis, CA 95616–5295, USA, and bDepartment of Chemistry, University of Kentucky, Lexington, Kentucky, USA
Correspondence e-mail:

10.1.2. Beneficial effects of low temperature

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Biocrystals near room temperature are sensitive to X-rays and generally suffer radiation damage during data measurement. Often this damage is so rapid and severe that a number of different crystals are needed for a full data set. On occasion, damage is so rapid that data collection is impossible. Crystal decay is typically accompanied by changes in reflection profiles and cell dimensions, which alter the positions of diffraction maxima, exacerbating the problem of changing diffraction intensities. The use of more than one crystal invariably introduces inaccuracies. Intensities from a crystal near the end of its usable life will have decay errors. Individual samples of biocrystals frequently have measurable differences in structure; merging of data will result in an average of the structures encountered, with concomitant loss of definition. Crystals cooled to near liquid-N2 temperature typically show a greatly reduced rate of radiation damage, often to the extent that it is no longer an issue of major concern. The protection from radiation damage was noted early on (Haas & Rossman, 1970[link]) and numerous cases were observed by Petsko (1975)[link]. A noteworthy example is the successful prevention of radiation damage to crystals of ribosome particles (Hope et al., 1989[link]).

Radiation damage appears to be most commonly initiated by photoelectrons and propagated by their inelastic scattering from nearby atoms, creating positively charged species and a cascade of secondary electrons (Garman & Nave, 2009[link]). These can further interact with either protein or with water to form reactive, charged species and free radicals. At sufficiently low temperature, two effects can influence the rate of damage: movement of the reactive species is impeded and the activation energy for reaction is not available. A revealing observation has been described by Hope (1990)[link], where a crystal that had been exposed to syn­chrotron radiation for many hours at 85 K showed no overt signs of radiation damage, but as the crystal was being warmed toward room temperature, it suddenly turned black and curled up like a drying leaf. More commonly, crystals turn yellow under X-ray irradiation, and bubbles and cracks appear on warming. The rate of free-radical formation would be little affected by temperature, so that when sufficient mobility and activation energy become available, the stored radicals will react.

There is a general consensus that radiation damage, even in high-flux synchrotron beams, can be slowed by cooling to liquid-helium temperatures. The extent of radiation damage is expected to depend on the nature of the macromolecule, and literature examples clearly illustrate this. Meents et al. (2007)[link] have reported an extensive study of insulin and holoferritin at 15 and 90 K. They observed statistically significant but relatively small reductions in radiation damage; for holoferritin, 23% less damage at the lower temperature, and for insulin about 6%. In a study of Streptomyces rubiginosus D-xylose isomerase, Chinte et al. (2007)[link] found a lifetime extension of 25% at 8 K compared to data collection at 100 K. Corbett et al. (2007)[link] compared results from data collected at ~40 and at 110 K in a study of the metalloprotein putida­redoxin. They report that radiation-induced photoreduction at 110 K resulted in misleading structure interpretation. At 40 K the photoreduction did not occur. The damage mechanism in this case is related to a change in oxidation state of a central metal atom. The authors made a strong argument for measuring data for metalloproteins at the lowest possible temperature. Chinte et al. (2007)[link] pointed out that the additional cost of liquid-helium-temperature measurements compared to 90–100 K measurements is small, and that the advantages can significantly outweigh the increased cost.

In recent years, the ability to calibrate X-ray beam intensity, to calculate reasonable approximations to accumulated radiation dose and to assess crystal degradation by means of various spectroscopies (e.g. UV–visible, IR, Raman, XAFS, XANES) in concert with diffraction has brought dramatic advances in understanding radiation damage by X-rays. A concise account has been given by Garman & Nave (2009)[link]. Useful suggestions for avoidance of radiation damage, including cases where multiple crystals are required, have been given by Holton (2009)[link]. Briefly, knowledge of beamline photon flux density (photons µm−2 s−1), the dose ratio (a function of crystal composition and X-ray wavelength), the crystal shape and the cross section intensity profile of a given X-ray beam allows an estimate of the maximum tolerable exposure time for a particular crystal. Mechanical stability of the crystal mount

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The mechanical stability of samples is also of concern. Crystals mounted in capillaries and kept wet are prone to movement, giving rise to difficulties with intensity measurements. A crystal at cryotemperature is rigidly attached to its mount; slippage is impossible. Effect on resolution

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The effects on radiation damage and mechanical stability are clear-cut, and provide the main reasons for using cryotechniques. Resolution can also be affected, but the connection between temperature and resolution is neither simple nor obvious.

In any crystal, the Boltzmann distribution law is an important factor in determining the accuracy of the replication of structure from one unit cell to another. For many small-molecule crystals, just one arrangement corresponds to a distinct energy minimum. The result is a well ordered structure. With macromolecules, the typical situation is one where a number of arrangements correspond to similar energies. Accordingly, a number of atomic arrangements will be expressed in the crystal. Although the relative values of local minima depend on the temperature, one cannot count on a significant change in ordering by cooling the crystal. Instead, some distribution will be frozen in.

If poor resolution is the result of rapid radiation damage, data collection at cryotemperature can lead to much improved resolution. However, if poor resolution is caused mainly by inexact replication from one unit cell to another, lowering the temperature may have little effect on resolution. If the mosaic spread in the crystal increases upon cooling, resolution may even deteriorate.

In a model proposed by Hope (1988[link]), a relationship between resolution r and temperature T is given by [r_{2} = r_{1}[(B_{0} + bT_{2})/(B_{0} + bT_{1})]^{1/2}.]Here [r_{1}] is the resolution at [T_{1}], [r_{2}] is the resolution at [T_{2}], [B_{0}] is the value of B at [T = 0] and b is a proportionality constant. There are two underlying assumptions: (1) the overall atomic distribution does not change significantly with temperature and (2) for any given T, the temperature factor [i.e. [\exp(-B\sin^{2}\theta/\lambda^{2})]] at the resolution limit has the same value; thus the effects of scattering factors and Lorentz–polarization factors are ignored. We see that if [B_{0}] is the predominant term, lowering T will not have much effect, whereas for small [B_{0}] (a relatively well ordered structure) the effect of T on r can be large. For example, if the room-temperature resolution is 1.5 Å, the resolution at 100 K can be around 1 Å, but if the room-temperature resolution is around 3 or 4 Å, little change can be expected. A qualitative assessment of these effects was clearly stated by Petsko (1975)[link]. Annealing of biocrystals

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Prior to about 1996, it was thought that thawing of a flash-cooled crystal would inevitably lead to its demise. In spite of anecdotal evidence that some biocrystals could survive warming and re-cooling, this notion persisted until work by Harp et al. (1998)[link] and by Yeh & Hol (1998)[link] showed that some biocrystals could be annealed under certain well defined conditions. The method of Harp et al. (1998)[link] involved transfer of a flash-cooled crystal from the cold stream to a cryoprotective solution at room temperature for 3 min followed by a second flash cooling. The Yeh & Hol (1998)[link] technique, on the other hand, is performed in situ by simply blocking the cold stream for 1–2 s (i.e. until melting is observed), after which time the blockage is removed to re-cool the sample. Both these annealing protocols were shown to be capable of dramatic improvements in diffraction quality, both in terms of reduced mosaicity and improved resolution. A plausible mechanism involving the release of cooling-induced lattice stress by defect migration and solvent transport was suggested by Kriminski et al. (2002)[link]. Other work (Parkin & Hope, 2003[link]; Juers & Matthews, 2004[link]; Weik et al., 2005[link]) supports the notion of solvent transport, possibly as a result of solvent crystallization (Weik et al., 2001[link]) or other phase transition (Parkin & Hope, 2003[link]) in the aqueous regions within biocrystals. Additional benefits from sub-77 K cooling with helium

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As is the case with nitrogen cooling, it is unlikely that thermo­dynamic equilibrium will be reached by cooling to liquid-helium temperatures. The change that will certainly take place on cooling to liquid-helium temperature is that true thermal motion will be greatly reduced. One result of this is that individual atom peaks will become much sharper. For example, electron-density maxima for well ordered atoms will increase by a factor of about three on cooling from 90 to 10 K. Potentially, this can allow a more detailed interpretation of a structure with a resolution limit better than about 1.5 Å, and also for the better ordered regions of a structure with poorer overall resolution. In general, however, it is not realistic to expect a significant resolution improvement in low-resolution structures based on the effects of temperature alone. Improvements related to diminished radiation damage, on the other hand, can be significant. Two studies illustrate the effects discussed here.

The effects of helium cooling on a high-resolution structure are well illustrated in a study by Petrova et al. (2006)[link]. They studied a complex of human aldose reductase at 15, 60 and 100 K. The complex has yielded data to 0.66 Å resolution, and thus represents a generally highly ordered structure. The emphasis of the study was on the behaviour of the atomic displacement parameters (ADPs). It was found that the major ADP component for well ordered atoms is temperature driven, as it would be in normal small-molecule structures. A large proportion of the atoms at 15 K have B values of 2 Å2 or less (about 0.025 Å2 or less in terms of U values). At 100 K, the corresponding cutoff is about 5 Å2. Cooling to 15 K allows large portions of the structure to be determined with a precision that would be considered excellent for small molecules. However, the average isotropic B value for the `best' Cα atoms at 15 K is still 3.9 Å2 (a U value of about 0.05 Å2). This indicates that the positional parameters for many of these atoms in reality are composites of closely spaced positions. The best average protein ADPs at 15 K are about the magnitude of small-molecule ADPs at room temperature. This sets unfortunate limits to the attainable accuracy of structural and electron-density parameters.

Hexagonal hen egg-white lysozyme has a relatively well ordered structure, but there are significant regions with multiple conformations. Brinkmann et al. (2006)[link] measured diffraction data at 10 K to a resolution limit of 1.46 Å. The results indicate that major areas of disorder are present, illustrating that structural disorder persists at the lowest temperatures.

Although helium is more expensive than nitrogen as a coolant, the added cost for a helium-temperature data set is usually trivial. Equipment design and operating methods have developed to a stage where there is no significant operational difference between nitrogen and helium cooling when manual crystal handling is used.


Parkin, S. & Hope, H. (2003). Low-temperature water reconstruction in concanavalin A, with implications for controlled protein crystal annealing. Acta Cryst. D59, 2228–2236.
Brinkmann, C., Weiss, M. S. & Weckert, E. (2006). The structure of the hexagonal crystal form of hen egg-white lysozyme. Acta Cryst. D62, 349–355.
Chinte, U., Shah, B., Chen, Y.-S., Pinkerton, A. A., Schall, C. A. & Hanson, B. L. (2007). Cryogenic (<20 K) helium cooling mitigates radiation damage to protein crystals. Acta Cryst. D63, 486–492.
Corbett, M. C., Latimer, M. J., Poulos, T. L., Sevrioukova, I. F., Hodgson, K. O. & Hedman, B. (2007). Photoreduction of the active site of the metalloprotein putidaredoxin by synchrotron radiation. Acta Cryst. D63, 951–960.
Garman, E. F. & Nave, C. (2009). Synchrotron radiation damage in protein crystals examined under various conditions by different methods. J. Synchrotron Rad. 16, 129–132.
Haas, D. J. & Rossmann, M. G. (1970). Crystallographic studies on lactate dehydrogenase at −75 °C. Acta Cryst. B26, 998–1004.
Harp, J. M., Timm, D. E. & Bunick, G. J. (1998). Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography. Acta Cryst. D54, 622–628.
Holton, J. M. (2009). A beginner's guide to radiation damage. J. Synchrotron Rad. 16, 133–142.
Hope, H. (1988). Cryocrystallography of biological macromolecules: a generally applicable method. Acta Cryst. B44, 22–26.
Hope, H. (1990). Cryocrystallography of biological macromolecules at ultra-low temperature. Annu. Rev. Biophys. Biophys. Chem. 19, 107–126.
Hope, H., Frolow, F., von Böhlen, K., Makowski, I., Kratky, C., Halfon, Y., Danz, H., Webster, P., Bartels, K. S., Wittmann, H. G. & Yonath, A. (1989). Cryocrystallography of ribosomal particles. Acta Cryst. B45, 190–199.
Juers, D. H. & Matthews, B. W. (2004). The role of solvent transport in cryo-annealing of macromolecular crystals. Acta Cryst. D60, 412–421.
Kriminski, S., Caylor, C. L., Nonato, M. C., Finkelstein, K. D. & Thorne, R. E. (2002). Flash-cooling and annealing of protein crystals. Acta Cryst. D58, 459–471.
Meents, A., Wagner, A., Schneider, R., Pradervand, C., Pohl, E. & Schulze-Briese, C. (2007). Reduction of X-ray-induced radiation damage of macromolecular crystals by data collection at 15 K: a systematic study. Acta Cryst. D63, 302–309.
Petrova, T., Ginell, S., Mitschler, A., Hazemann, I., Schneider, T., Cousido, A., Lunin, V. Y., Joachimiak, A. & Podjarny, A. (2006). Ultrahigh-resolution study of protein atomic displacement parameters at cryotemperatures obtained with a helium cryostat. Acta Cryst. D62, 1535–1544.
Petsko, G. A. (1975). Protein crystallography at sub-zero temperatures: cryoprotective mother liquors for protein crystals. J. Mol. Biol. 96, 381–392.
Weik, M., Kryger, G., Schreurs, A. M. M., Bouma, B., Silman, I., Sussman, J. L., Gros, P. & Kroon, J. (2001). Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures. Acta Cryst. D57, 566–573.
Weik, M., Schreurs, A. M. M., Leiros, H.-K. S., Zaccai, G., Ravelli, R. B. G. & Gros, P. (2005). Supercooled liquid-like solvent in trypsin crystals: implications for crystal annealing and temperature-controlled X-ray radiation damage studies. J. Synchrotron Rad. 12, 310–317.
Yeh, J. I. & Hol, W. G. J. (1998). A flash-annealing technique to improve diffraction limits and lower mosaicity in crystals of glycerol kinase. Acta Cryst. D54, 479–480.

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