Crystallization of macromolecules

For application of X-ray diffraction to a macromolecule, the protein or nucleic acid must first be crystallized. Not only must crystals be grown, but they must be reasonably large, high-quality crystals that are suitable for a high-resolution X-ray diffraction study. The crystallization step has emerged as the primary obstacle in macromolecular crystallography. This is principally due to the empirical nature of the methods employed to overcome it, and the complexity of the molecules involved (McPherson, 1976, 1982, 1999). 

Macromolecules are intricate physical-chemical systems whose properties vary as a function of environmental influences such as temperature, pH, ionic strength, contaminants, and solvent composition, to name only a few. They are structurally dynamic, often microhetero-geneous, aggregating systems, and they change conformation in the presence of ligands. Superimposed on this is the limited nature of our current understanding of macromolecular crystallization phenomena and the forces that promote and maintain protein and nucleic acid crystals. 

Introduction to Macromolecular Crystallography, Second Edition By Alexander McPherson Copyright 2009 John Wiley Sons, Inc. 

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The latest results of a controlled crystallization of macromolecules are the polymer fibrids which are a completely new modification of synthetic polymers as far as the micro- and macro-structure is concerned. They exist of small fibers having a length of up to some millimeters, which are highly oriented, and which have a macro-morphology similar to that of cellulose pulp. 

Garman, E. F (1991). Modern methods for rapid X-ray diffraction data collection from crystals of macromolecules. In Methods in Molecular Biology, vol. 56, Crystallographic Methods and Protocols, Jones, C., MuUoy B. and Sanderson, M. R., eds. Humana Press. 

Alexander McPherson and Paul J. Shlichta have suggested using insoluble minerals as heterogeneous nuclei for the crystallization of macromolecules. They obtained excellent protein crystals, which could be cleaved from the mineral nucleus and used for X-ray diffraction studies. The mineral is introduced into a supersaturated solution of the material to be crystallized. As supersaturation increases, nucleation occurs on a specific face of the mineral nucleus, and a crystal begins to grow. The orientation and periodicity of the molecules on the nucleus surface promote an oriented overgrowth that has a similar periodicity. 

The perplexing difficulties that arise in the crystallization of macromolecules, in comparison with conventional small molecules, stem from the greater complexity, lability, and dynamic properties of proteins and nucleic acids. The description offered above of labile and metastable regions of supersaturation are still applicable to macromolecules, but it must now be borne in mind that as conditions are adjusted to transport the solution away from equilibrium by alteration of its physical and chemical properties, the very nature of the solute molecules is changing as well. As temperature, pH, pressure, or solvation are changed, so may be the conformation, charge state, or size of the solute macromolecules. 

Crystals of macromolecules, like those shown in Figure 3.1, are like crystals of all other kinds. They are precisely ordered three-dimensional arrays of molecules that may be characterized by a concise set of determinants that exactly define the disposition and periodicity of the fundamental units of which they are composed. The set of parameters is comprised of three elements. These define the symmetry properties, the repetitive and periodic features, and the distribution of atoms in the repeating unit. The properties may be separated and understood by considering how a crystal can be developed as a three-dimensional form from a basic building block (the asymmetric unit), by the application of symmetry (the space group), and translation (the unit cell, or lattice). As illustrated in Figure 3.2, this can be accomplished in four stages. 

McPherson, A. 1985. Crystallization of macromolecules general principles. Meth. Enzymol. 114 112-120. 

Simultaneous polymerization emd crystallization is another approach to memroscopic, defect-free single crystals of macromolecules (59). Recent examples include a preparative method for mixed metal coordination polymers (60), emd M. Hemack emd coworkers have reacted hemipotphyreizine (6 with iron (II) acetate in nitrobenzene to obtain single crystals of em oxygen-bridg polymer with iron in a -i- 4 oxidation state. 

As remarked in note (3) of Table 2.1 crystals of macromolecules cannot contain an inversion centre, a mirror or a glide plane. The diffraction pattern from a protein crystal can, however, contain an inversion centre (otherwise known as a centre of symmetry and a mirror plane. The symmetry symbols given here are for those symmetry elements seen therefore for macromolecular crystals and their diffraction patterns. 

Matsushige K, Takemura T (1980) Crystallization of macromolecules under high pressure. J. Crystal Growth 48 343... 

K. A. Mauritz, E. Baer, A. J. Hopfinger, The Epitaxial Crystallization of Macromolecules,... 

Let us point out that according to Wunderlich [2], plastic crystals may also be considered as mesophases. They are characterized by positional order but orientational disorder of the structural motif. Molecules of plastic crystals are generally close to spherical so that there is no high-energy barrier to their reorientation. Of course, the condition of a spherical shape of the molecules may not be fulfilled by the macromolecular chains of linear synthetic polymers, which generally crystallize in extended chains or helical conformations [2]. However, there is at least one case of crystals of macromolecules presenting orientational disorder of the structural motif as in plastic crystals. 

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