The beauty of crystals can be found in both the naturally appearing minerals such as diamonds or quartzite crystals and the industrial products such as sugar and protein crystals. A consequence of well-defined arrangement of the building blocks is the outer shape of the crystals, which are limited by flat faces that intersect under well-defined angles determined by the lattice. A further equally important consequence of packing is the well-known purification during crystallization; only molecules of one type are incorporated, while most other molecules are rejected by the growing interface.
Crystallization belongs to the oldest unit operations known to mankind. Today, crystalline products can be found in every aspect of life. Relevant product properties are determined by crystal properties and thus tailored via crystallization. The applications of crystallization in industry range from the isolation of the few milligrams of a substance newly synthesized in the laboratory – where a well-defined melting point is used to both achieve and prove a decent purity of the crop and as an identity check – to a mass crystallization carried out in every diverse industry.
Figure 1. Protein crystals and structure
Pipeline for Protein Crystallography
1. Protein expression and purification
To start a protein crystallography project, we first need to obtain well-diffracted crystals of the protein sample. Getting homogeneous protein samples with high purity (above 95%) and enough quantity (a few milligrams) is essential to the nucleation and growth of crystals. After expressing the protein using your preferable host system, purification is usually performed in a stepwise procedure with affinity, ion-exchange and size-exclusion chromatography. For quality check, one or more biophysical methods are commonly employed, including analytical ultracentrifugation, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), differential scanning fluorimetry (DSF) and CD spectroscopy. One should keep in mind that proper and detailed characterization of a protein preparation in crystallography is much more important than in biochemical experiments, where bioactivity and partial purity are sufficient criteria for the quality of the preparation.
2. Protein crystallization
Crystallization is a process governed by both thermodynamic and kinetic factors, in which molecules arrange themselves in a natural repetitive style to form a well-packing three-dimensional reticulum that is called crystal. The crystallization process consists of two major events: nucleation and crystal growth. Nucleation is the step where the molecules dispersed in the solvent start to gather into nanometer-scale clusters and become stable under the operating conditions. Unstable clusters will re-dissolve into solution, and the stable ones constitute the nuclei when they reach a critical size that can be affected by temperature, supersaturation, etc. It is at the stage of nucleation that the atoms arrange in a defined and periodic manner which defines the crystal structure — note that "crystal structure" is a special term that refers to the relative arrangement of the atoms, not the macroscopic properties of the crystal (size and shape), although those are an outcome of the internal crystal structure.
Figure 2. The different areas of a protein-precipitant equilibrium in terms of the concentrations of both components. (Areas shown in yellow (a) and white (b) represent the conditions under which the protein is in solution. The areas depicted in blue represent the conditions under which the protein appears as a precipitate. Both areas are separated by another area (shown in pink) with some supersaturation conditions, suitable for nucleation and crystal growth.)
3. Protein structure determination and refinement
Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract X-rays efficiently while withstanding radiation damage. We can obtain high-resolution structural information from microcrystals (less than 1 mm x 1 mm x 3 mm) of the well-characterized protein samples.
4. Protein characterization
Biophysical characterization of proteins in developing biopharmaceuticals is concerned with the analysis and characterization of the higher-order structure (HOS) or conformation of protein based drugs. Starting from the very basics of protein structure, we are on a journey on how to best achieve this goal using the key relevant and practical methods commonly employed in the biopharmaceutical industry today as well as up and coming promising methods that are now gaining increasing popularity.
Creative Biostructure can also provide various of services based on the UniCrys™ Crystallography Platform. Please see the Related Sections for detail information and feel free to contact us for a detailed quote.