Macromolecular crystallography

Single-crystal X-ray crystallographic analysis of proteins is the method of choice for obtaining three-dimensional structural information. Crystallization trials are carried out in very small scale (e.g., 200 nL of protein solution) so that a very large number of different crystallization conditions (e.g. more than 3000) can be tested with a relatively small (< 1 mL) volume of protein solution at ca. 10 mg/mL.

 

Once protein crystals (typically << 1 mm3 in size) have been obtained, X-ray diffraction measurements can be carried out, either with in-house equipment or at a synchrotron radiation source. The three-dimensional structure is initially obtained in the form of an experimental electron density map and that map is interpreted, resulting in an atomic model for the protein structure. Once an atomic model is known, crystals of the protein of interest with potential drugs can be prepared and analyzed in a straightforward fashion.

 

Biomolecular NMR

As an alternative to X-ray crystallography in cases where small (up to 20 kDa) target proteins fail to crystallize, structural NMR is a good alternative. Crystallization is not required, but labelled samples (15N and 13C) have to be prepared in addition to the native protein. The structural information is obtained from several NMR spectra measured using different protocols, and results in a family of closely-related structural models.

The main advantage of NMR is that it works in solution, with experimental conditions much closer to real life. The main disadvantages of NMR are that the sample preparation can be much more expensive and that data interpretation is much more complex and time-consuming than X-ray crystallography.

NMR spectroscopy can also be used for drug screening and hit optimization, mapping of protein-ligand interactions during drug discovery process, structure-activity relationships and studies of protein-protein complexes. It is also useful for protein quality control and as a tool for fast identification of proteins suitable for crystallization.

 

Molecular modelling

Molecular modelling techniques can go beyond and complement the structural information obtained by experimental techniques. Structure prediction of small and large molecules, molecular dynamics simulation of biomolecules, molecular docking, continuum electrostatics and molecular graphics are examples of techniques that can be applied to understand biological molecules, their properties and their interactions with other molecules.

 

ABC transporter

 

Hemagglutinin