Crystallisation of Proteins and Macromolecular Complexes: Past, Present and Future
Authors: Deller Marc C , Rupp Bernhard 
Published: August 2014

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.