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

International Tables for Crystallography (2006). Vol. F, ch. 4.1, pp. 86-88   | 1 | 2 |

Section 4.1.3. Parameters that affect crystallization of macromolecules

R. Giegéa* and A. McPhersonb

aUnité Propre de Recherche du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg CEDEX, France, and bDepartment of Molecular Biology & Biochemistry, University of California at Irvine, Irvine, CA 92717, USA
Correspondence e-mail:

4.1.3. Parameters that affect crystallization of macromolecules

| top | pdf | Crystallizing agents

| top | pdf |

Crystallizing agents for macromolecules fall into four categories: salts, organic solvents, long-chain polymers, and low-molecular-mass polymers and non-volatile organic compounds (McPherson, 1990[link]). The first two classes are typified by ammonium sulfate and ethanol; higher polymers, such as PEG 4000, are characteristic of the third. In the fourth are placed compounds such as MPD and low-molecular-mass PEGs. A compilation of crystallizing agents and their rates of success, as taken from the CARB/NIST database (Gilliland et al., 1994[link]), is presented in Table[link].

Salts exert their effects by dehydrating proteins through competition for water molecules (Green & Hughes, 1955[link]). Their ability to do this is roughly proportional to the square of the valences of the ionic species composing the salt. Thus multivalent ions, particularly anions, are the most efficient. One might think there would be little variation between different salts, so long as their ionic valences were the same, or that there would be little variation between two different sulfates, such as Li2SO4 and (NH4)2SO4. This, however, is often not the case. In addition to salting out (a dehydration effect) or lowering the chemical activity of water, there are specific protein–ion interactions that have other consequences (Riès-Kautt & Ducruix, 1991[link], 1999[link]). These result from the polyvalent character of individual proteins, their structural complexity, and the dependence of their physical properties on environmental conditions and interacting molecules. It is never sufficient, therefore, when attempting to crystallize a protein to examine only one or two salts and ignore a broader range. Changes in salt sometimes produce crystals of varied quality, morphology and diffraction properties.

It is usually not possible to predict the molarity of a salt required to crystallize a particular protein without some prior knowledge of its behaviour. In general, it is a concentration just a few per cent less than that which yields an amorphous precipitate. To determine the precipitation point with a particular agent, a 10 µl droplet of a 5–15 mg ml−1 macromolecule solution is placed in the well of a depression slide and observed under a microscope as increasing amounts of salt solution or organic solvent (in 1–2 µl increments) are added. If the well is sealed between additions with a cover slip, the increases can be made over a period of many hours. Indeed, the droplet should equilibrate for 10–30 min after each addition, or longer in the neighbourhood of the precipitation point.

The most common organic solvents used are ethanol, methanol, acetone and MPD. They have been frequently used for crystallizing nucleic acids, particularly tRNAs and duplex oligonucleotides (Dock et al., 1984[link]; Dock-Bregeon et al., 1999[link]). This, in part, stems from the greater tolerance of polynucleotides to organic solvents and their polyanionic character, which appears to be more sensitive to dielectric effects than proteins. Organic solvents should be used at low temperature (especially when volatile), and should be added slowly and with good mixing.

PEGs are polymers of various length that are useful in crystallogenesis (McPherson, 1976[link]; Table[link]). The low-molecular-mass species are oily liquids, while those with [M_{r} \gt 1000] exist as either waxy solids or powders at room temperature. The sizes specified by manufacturers are mean [M_{r}] values and the distributions around these means vary. In addition to volume-exclusion properties, PEGs share characteristics with salts that compete for water and produce dehydration, and with organic solvents that reduce the dielectric properties of the medium. PEGs also have the advantage of being effective at minimal ionic strength and providing low-electron-density media. The first feature is important because it provides better affinities for ligand binding than do high-ionic-strength media. Thus, there is greater ease in obtaining heavy-atom derivatives and in forming protein–ligand complexes. The second characteristic, their low electron density, implies a lower noise level for structures derived by X-ray diffraction.

The most useful PEGs in crystallogenesis are those in the range 2000–6000. Sizes are not generally completely interchangeable for a given protein, and thus this parameter has to be optimized by empirical means. An advantage of PEG over other agents is that most proteins crystallize within a rather narrow range of PEG concentration (~4–18%). In addition, the exact PEG concentration at which crystals form is rather insensitive, and if one is within 2–3% of the optimal value, some success will be achieved. The advantage is that when conducting initial trials, one can use a fairly coarse selection of concentrations. This means fewer trials with a corresponding reduction in the amount of material expended.

Since PEG is not volatile, this agent must be used like salt and equilibrated with the protein by dialysis, slow mixing, or vapour diffusion. This latter approach has proved the most popular. When the reservoir concentration is in the range 5–12%, the protein solution to be equilibrated should be at an initial concentration of about half that. When the target PEG concentration is higher than 12%, it is advisable to initiate the equilibration at no more than 4–5% below the final value. This reduces time lags during which the protein might denature. Crystallization of proteins with PEG has proved most successful when ionic strength is low, and most difficult when high. If crystallization proceeds too rapidly, addition of some neutral salt may be used to slow growth. PEG can be used over the entire pH range and a broad temperature range. It should be noted that solutions with PEG may serve as media for microbes, particularly moulds, and if crystallization is attempted at room temperature or over extended periods of time, then retardants, such as azide ([\sim\! 0.1\]%), must be included in the protein solutions. Other chemical, physical and biochemical variables

| top | pdf |

Many physical, chemical and biological variables influence, to a greater or lesser extent, the crystallization of macromolecules (Table[link]). The difficulty in arriving at a just assignment of importance for each factor is substantial for several reasons. Every protein (or nucleic acid) is different in its properties, and this even applies to proteins that differ by no more than one or a few amino acids. In addition, each factor may differ in importance. Because of that, there are few means available to predict, in advance, the specific values of a variable or sets of conditions that might be most profitably explored. Furthermore, the various parameters under control are not independent and their interrelations may be difficult to discern. Thus, it is not easy to give firm guidelines regarding physical or chemical factors that can increase the probability of success in crystallizing a particular macromolecule. Among physical parameters, only temperature and pH have been studied carefully; for pressure or magnetic and electric fields, rather few investigations have been carried out (see above), and virtually nothing is known about the effects of sound, vibrations or viscosity on the growth or final quality of protein crystals.

Temperature may be of great importance or it may have little bearing at all. In general, it is wise to conduct parallel investigations at 4 and 20 °C. Even if no crystals are observed at either temperature, differences in the solubility behaviour of a protein with different crystallizing agents and with various effector molecules may give some indication as to whether temperature is likely to play an important role (Christopher et al., 1998[link]). Generally, the solubility of a protein is more sensitive to temperature at low ionic strength than at high. One must remember, however, that diffusion rates are less, and equilibration occurs more slowly, at lower than higher temperatures, so the time required for crystal formation may be longer at lower temperatures. Although most crystallization trials are done at low (∼4 °C) or medium (∼20 °C) temperatures, higher temperatures in the range 35–40 °C should not be ignored, particularly for molecules that tend to aggregate and for nucleic acids (Dock-Bregeon et al., 1988[link]).

Another important variable is pH. This is because the charge character of a macromolecule and all of its consequences are intimately dependent on the ionization state of its components. Not only does its net charge change with pH (and the charge distribution), but so do its dipole moment, conformation and often its aggregation state. Thus, an investigation of the behaviour of a specific macromolecule as a function of pH is an essential analysis that should be carried out in performing crystallization assays. As with temperature, the procedure is first to conduct trials at coarse intervals over a broad pH range and then to refine trials in the neighbourhoods of those that showed promise. In refining the pH for optimal growth, it should be recalled that the difference between amorphous precipitate, microcrystals and large single crystals may be only a ΔpH of no more than 0.5. Additives

| top | pdf |

Intriguing questions with regard to optimizing crystallization conditions concern which additional compounds should comprise the mother liquor in addition to solvent, macromolecule and crystallizing agent (Sauter, Ng et al., 1999[link]). Polyamines and metal ions are useful for nucleic acids. Some useful effectors for proteins are those that maintain their structure in a single, homogeneous and invariant state (Timasheff & Arakawa, 1988[link]; Sousa et al., 1991[link]). Such effectors, sometimes named cosmotropes (Jerusalmi & Steitz, 1997[link]), are polyhydric alcohols, like glycerol, sugars, amino acids or methylamino acids. Sulfobetaines also show remarkable properties (Vuillard et al., 1994[link]). Reducing agents, like glutathione or 2-mercaptoethanol, which prevent oxidation, may be important additives, as may chelating compounds, like EDTA, which protect proteins from heavy- or transition-metal ions. Inclusion of these compounds may be desirable when crystallization requires a long period of time to reach completion. When crystallization is carried out at room temperature in PEG or in low-ionic-strength solutions, the growth of microbes that may secrete enzymes that can alter the integrity of the macromolecule under study must be prevented (see below).

Substrates, coenzymes and inhibitors can fix a macromolecule in a more compact and stable form. Thus, a greater degree of structural homogeneity may be imparted to a population of macromolecules by complexing them with a natural ligand before attempting crystallization. In terms of crystallization, complexes have to be treated as almost entirely separate problems. This may permit a new opportunity for growing crystals if the native molecule is obstinate. Just as natural substrates or inhibitors are often useful, they can also have the opposite effect of obstructing crystal formation. In such cases, care must be taken to eliminate them from the mother liquor and from the purified protein before crystallization is attempted. Finally, it should be noted that the use of inhibitors or other ligands may sometimes be invoked to obtain a crystal form different from that grown from the native protein.


Christopher, G. K., Phipps, A. G. & Gray, R. J. (1998). Temperature-dependent solubility of selected proteins. J. Cryst. Growth, 191, 820–826.Google Scholar
Dock, A.-C., Lorber, B., Moras, D., Pixa, G., Thierry, J.-C. & Giegé, R. (1984). Crystallization of transfer ribonucleic acids. Biochimie, 66, 179–201.Google Scholar
Dock-Bregeon, A.-C., Chevrier, B., Podjarny, A., Moras, D., deBear, J. S., Gough, G. R., Gilham, P. T. & Johnson, J. E. (1988). High resolution structure of the RNA duplex [U(U–A)6A]2. Nature (London), 209, 375–378.Google Scholar
Dock-Bregeon, A.-C., Moras, D. & Giegé, R. (1999). Nucleic acids and their complexes. In Crystallization of nucleic acids and proteins, 2nd ed. A. Ducruix & R. Giegé, edited by Oxford University Press.Google Scholar
Gilliland, G., Tung, M., Blakeslee, D. M. & Ladner, J. E. (1994). Biological macromolecule crystallization database, version 3.0: new features, data and the NASA archive for protein crystal growth data. Acta Cryst. D50, 408–413.Google Scholar
Green, A. A. & Hughes, W. L. (1995). Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents. Methods Enzymol. 1, 67–90.Google Scholar
Jerusalmi, D. & Steitz, T. A. (1997). Use of organic cosmotropic solutes to crystallize flexible proteins: application to T7 RNA polymerase and its complex with the inhibitor T7 lysozyme. J. Mol. Biol. 274, 748–756.Google Scholar
McPherson, A. (1976). Crystallization of proteins from polyethylene glycol. J. Biol. Chem. 251, 6300–6303.Google Scholar
McPherson, A. (1990). Current approaches to macromolecular crystallization. Eur. J. Biochem. 189, 1–23.Google Scholar
Riès-Kautt, M. & Ducruix, A. (1991). Crystallization of basic proteins by ion pairing. J. Cryst. Growth, 110, 20–25.Google Scholar
Riès-Kautt, M. & Ducruix, A. (1999). Phase diagrams. In Crystallization of nucleic acids and proteins, edited by A. Ducruix & R. Giegé, 2nd ed. Oxford University Press.Google Scholar
Sauter, C., Ng, J. D., Lorber, B., Keith, G., Brion, P., Hosseini, M. W., Lehn, J.-M. & Giegé, R. (1999). Additives for the crystallization of proteins and nucleic acids. J. Cryst. Growth, 196, 365–376.Google Scholar
Sousa, R., Lafer, E. M. & Wang, B.-C. (1991). Preparation of crystals of T7 RNA polymerase suitable for high resolution X-ray structure analysis. J. Cryst. Growth, 110, 237–246.Google Scholar
Timasheff, S. N. & Arakawa, T. (1988). Mechanism of protein precipitation and stabilization by co-solvents. J. Cryst. Growth, 90, 39–46.Google Scholar
Vuillard, L., Rabilloud, T., Leberman, R., Berthet-Colominas, C. & Cusack, S. (1994). A new additive for protein crystallization. FEBS Lett. 353, 294–296.Google Scholar

to end of page
to top of page