Crystallisation of Proteins and Macromolecular Complexes: Past, Present and Future

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Crystallisation of Proteins and Macromolecular Complexes: Past, Present and Future

Marc C Deller, The Scripps Research Institute, La Jolla, California
Bernhard Rupp, Innsbruck Medical University, Innsbruck, Austria

Published online: August 2014

DOI: 10.1002/9780470015902.a0002718.pub2

Full Article on Wiley Online Library

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Abstract

The crystallisation of proteins and macromolecular complexes is an essential step for studying the three‐dimensional structure of the molecular components that make up living cells. Protein crystals are formed by the self‐assembly of protein molecules into an ordered, periodic lattice arrangement. Crystallisation is initiated by reducing the solubility of the protein sample by the addition of precipitating agents such as salts and polyethylene glycols (PEG). This creates a supersaturated solution from which a protein‐rich phase will eventually separate. Under favourable conditions, the protein‐rich phase can adopt the form of an ordered protein crystal. The most common technique used for crystallising proteins is vapour diffusion, which relies on the loss of water from the growth solution during equilibration with a reservoir of precipitant. Protein crystals can be extremely difficult to grow and often require exhaustive screening of many different parameters such as concentration and type of precipitant, pH, temperature, additives and variations in protein construct.

Key Concepts:

A detailed knowledge of the molecular structure of proteins and macromolecular complexes is essential for understanding how the molecular machinery of living cells operates.
Crystallography is the most prevalent method for studying the structures of proteins and macromolecular complexes at the heart of the cellular machinery.
Protein crystals are formed by the self‐assembly of large polypeptide chains into an ordered, regular and repeating lattice arrangement of protein molecules.
Crystallisation of proteins is generally carried out by the addition of precipitating agents to the protein solution, which results in the generation of a metastable, supersaturated solution from which a protein‐rich phase will eventually separate. Under favourable conditions, this protein‐rich phase can adopt the form an ordered protein crystal.
Several experimental techniques are available to crystallise proteins, including vapour diffusion, microbatch crystallisation under oil, microdialysis, free‐interface diffusion and crystallisation in Lipidic Cubic Phase (LCP).
The most commonly used crystallisation technique is vapour diffusion, which relies on the diffusion of water from the crystal growth solution to a precipitant reservoir.
Even the most beautiful protein crystals do not necessarily diffract X‐rays. Many protein crystals suffer from disordered regions as a result of thermal or static displacement of atoms. Disorder is often a result of flexible domains in the protein that may adversely affect how the protein molecules are packaged together in the crystal.
Recombinant DNA techniques can be used to engineer protein constructs that are more amenable to crystallisation by increasing the stability and/or solubility of the protein. Additionally, the amino acid residues on the surface of the protein can be modified to alter the intermolecular surface contacts within the crystal.
Complexes of proteins bound to small molecule substrates such as cofactors or potential drugs can be crystallised. Additionally, very large complexes between multiple proteins or with other macromolecules such as RNA or DNA can be crystallised.

Keywords: protein crystals; protein crystallography; protein crystallisation; X‐ray diffraction; structural biology; protein structure; macromolecular complexes

	Figure 1. The nature of protein crystals. (a) Protein crystals are formed by self‐organisation of protein molecules into an ordered, three‐dimensional network. In this example, the proteins are held together in a regular repeating fashion by a series of weak intermolecular interactions shown by the red, green and blue arrows. (b) Crystal of protein PC50891A grown in a nanodroplet that was setup by an automated robotics system. (c) Lysozyme crystal produced by hand pipetting of much larger volumes. Images of crystals acquired using a digital camera attached to an optical microscope at a magnification of approximately 80x. Adapted from Rupp ().
	Figure 2. Protein solubility diagram and nucleation process. The three main regions observed during the course of protein crystallisation are highlighted; stable protein solution (blue), metastable, supersaturated region (green) and unstable region (red). The spontaneous formation of crystallisation nuclei potentially leading to protein crystals occurs in the metastable region at higher supersaturation. Adapted from Rupp ().
	Figure 3. Common protein crystallisation methods. The most commonly employed methods include (a) hanging drop vapour diffusion and (b) sitting drop vapour diffusion. Hanging drop methods are typically employed for low‐throughput manual setups, whereas sitting drop methods are more amenable for robotics and automated setups. (c) Microbatch, (d) microdialysis and (e) free‐interface diffusion are less commonly used, but are powerful tools in the crystallographer's arsenal for proteins recalcitrant to crystallisation using standard techniques. (f) Microbatch Lipidic Cubic Phase (LCP) experiment used for crystallising membrane proteins. The LCP bolus is shown in red sandwiched between a glass cover slide and the crystallisation plate. (g) Workflow of a typical hanging drop vapour diffusion experiment. Adapted from Rupp ().
	Figure 4. Path of a successful vapour diffusion experiment through the protein solubility diagram. Example illustrates the principles of a typical hanging drop vapour diffusion experiment. Equal sized drops of protein (p; dark green circle) and crystallisation cocktail (c; blue circle) are pipetted onto a cover slide, inverted and sealed over a larger reservoir of crystallisation cocktail. This results in a starting drop (S, light‐green circle) with protein and crystallisation cocktail concentrations half that of the original (p/2 and c/2, respectively). As a result, water vapour diffuses out of the starting drop in an attempt to reestablish equilibrium. This gradually increases both the protein and precipitant concentrations in the drop, and once the solution is sufficiently supersaturated, nucleation of a crystal may occur (1) and if conditions are suitable, crystal growth may continue (2 through 4). Adapted from Rupp ().
	Figure 5. Increasing size and resolution range of macromolecular complexes in the PDB. Panel (a), (b) and (c) show the distribution of resolution, size and number of polymers for each year of PDB depositions, respectively. Some of the early PDB entries are shown in panel (d) along with some other structures of note from Table . Adapted from Jones ().

Article Title:	Crystallisation of Proteins and Macromolecular Complexes: Past, Present and Future
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