Introduction to protein crystallization

doi:10.1016/j.ymeth.2004.03.019

Methods

Volume 34, Issue 3, November 2004, Pages 254-265

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Abstract

Biological macromolecules can be crystallized by a variety of techniques, and using a wide range of reagents which produce supersaturated mother liquors. These may, in turn, be applied under different physical conditions such as temperature. The fundamental approaches to devising successful crystallization conditions and the factors that influence them are summarized here. For the Novice, it is hoped that this brief review might serve as a useful introduction and a stepping-stone to a successful X-ray strucutre determination. In addition, it may provide a framework in which to place the articles that follow.

Section snippets

Some history

Protein crystallization developed in the latter half of the 19th century for three reasons, (a) it provided a means for the purification of specific proteins from an otherwise impure mixture at a time when few other means existed, (b) it served as a demonstration that a protein had been purified (which even now is taken as a pretty good measure), and (c) it was an interesting laboratory curiosity. Initially, the crystallization of hemoglobin from a variety of sources was really nothing more

General approach

Macromolecular crystallization, which includes the crystallization of proteins, nucleic acids, and larger macromolecular assemblies such as viruses and ribosomes, is based on a rather diverse set of principles, experiences, and ideas. There is no comprehensive theory, or even a very good base of fundamental data, to guide our efforts, though that is being accumulated at this time. As a consequence, macromolecular crystal growth is largely empirical in nature, and demands patience, perseverance,

The nature of protein crystals

Macromolecular crystals like those seen in Fig. 1 are composed of approximately 50% solvent on average, though this may vary from 25 to 90% depending on the particular macromolecule. Protein or nucleic acid occupies the remaining volume so that the entire crystal is in many ways an ordered gel permeated by extensive interstitial spaces through which solvent and other small molecules freely diffuse.

In proportion to molecular mass, the number of bonds (salt bridges, hydrogen bonds, and

Screening and optimization

There are really two phases in the pursuit of protein crystals for an X-ray diffraction investigation, and these are (a) the identification of chemical, biochemical, and physical conditions that yield some crystalline material, though it may be entirely inadequate, and (b) the systematic alteration of those initial conditions by incremental amounts to obtain optimal samples for diffraction analysis. The first of these is fraught with the greater risk, as some proteins simply refuse to form

Supersaturation, nucleation, and growth

Crystallization of a molecule, or of any chemical species including proteins proceeds in two rather distinct but inseparable steps, nucleation and growth. Nucleation is the most difficult problem to address theoretically and experimentally because it represents a first order phase transition by which molecules pass from a wholly disordered state to an ordered one. Presumably this occurs through the formation of partially ordered or paracrystalline intermediates, in this case protein aggregates

Creating a state of supersaturation

In practice, one begins (with the exception of the batch method, see below) with a solution, a potential mother liquor, which contains some concentration of the protein below its solubility limit, or alternatively at its solubility maximum. The objective is then to alter matters so that the solubility of the protein in the sample is significantly reduced, thereby rendering the solution supersaturated. This may be done through several approaches, (a) altering the protein itself (e.g., by change

Methodology

The growth of protein crystals must be carried out in some physical apparatus that allows the investigator to alter the solubility of the protein or the properties of the mother liquor using one of the strategies in Table 1. Currently, these use almost exclusively microtechniques. Thus, crystallization “trials” with a particular matrix of conditions may be carried out with volumes of only a few microliters or less. Increasingly these employ plastic, multichambered trays for hanging and sitting

Precipitants

If one were to examine the reagents utilized in any of the commercial crystallization screens which are based on shotgun approaches, or examined the crystallization databases which have been compiled (see below), then it would become immediately apparent that a very wide range of precipitating (crystallizing) agents are used. Indeed, many agents have been employed usefully, and some, such as ammonium sulfate or polyethylene glycol, for a great number of successes. It is often necessary,

Factors affecting crystallization

There are many factors that affect the crystallization of macromolecules [34], [39] and many of these are summarized in Table 4. These may affect the probability of its occurring at all, the nucleation probability and rate, crystal growth rate, and the ultimate sizes and quality of the products. As noted above, pH and salt, or the concentrations of other precipitants are of great importance. The concentration of the macromolecule, which may vary from as low as 2 mg/ml to as much as 100 mg/ml, is

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Introduction to protein crystallization

Abstract

Section snippets

Some history

General approach

The nature of protein crystals

Screening and optimization

Supersaturation, nucleation, and growth

Creating a state of supersaturation

Methodology

Precipitants

Factors affecting crystallization

J. Struc. Biol.

J. Struc. Biol.

J. Struc. Biol.

J. Cryst. Growth

J. Cryst. Growth

J. Biol. Chem.

J. Cryst. Growth

J. Struc. Biol.

Methods Enzymol.

J. Struc. Biol.

J. Struct. Biol.

J. Struc. Biol.

Surface Sci

J. Cryst. Growth

J Cryst. Growth

Structure

Biochemistry and Biophysics Research Communications

Curr. Opin. Struct. Biol.

J. Cryst. Growth

J. Biol. Chem.

J. Biol. Chem.

Protein Crystallization: Techniques, Strategies and Tips

Nature

J. Am. Chem. Soc.

Crystallization of Nucleic Acids and Proteins, A Practical Approach