structural biology

CRYSTAL STRUCTURE DETERMINATION

COLLABORATION AND CONTRACT RESEARCH

Crystal structure determination consists of several stages. Also a degree of difficult varies considerably. People who are interested in collaborative projects or contract research should contact Juha Rouvinen for more detailed discussions.

Protein crystallography is a major method for experimental determination of three-dimensional structures of biological macromolecules. Typical features of protein crystal structures are

• precision: the positions of nonhydrogen atoms are determined typically at 0.1 Å accuracy. The accuracy is depending on the diffraction resolution.

• size: typical crystal structures of proteins contain thousands of atoms (MW 10 kDa to 1 MDa). Usually structure contains coordinates for nonhydrogen atoms. The position of hydrogen atoms can be experimentally determined at very high diffraction resolution.

• macromolecular complexes: crystal structure gives information about the quaternary structure and it is possible to determine protein-protein complex structures

• ligand complexes: it is possible to determine protein-ligand (such as receptor-drug) complex structures

• conformation: crystal structure normally represents one conformation. At high resolution some regions of the protein structure may show multiple conformations.

• water: protein crystals have a high solvent (water) content (30-70%). Therefore, crystal structures usually represent native structure: enzymes usually show activity in the crystal form. The crystal structure usually include ordered solvent molecules on the surface of the protein.

CRYSTALLIZATION

The prerequisite for crystal structure determination is the crystallization of protein. The goal is to get single ordered crystals which are able to diffract X-ray at high resolution. Resolution is depending especially on the order of molecules in the crystal, crystal size, and X-ray intensity.

Protein material for crystallization

• protein purity: protein is usually considered to be purely enough for crystallization trials if it shows a single band on the gel. Protein material is typically in a buffer solution without salt

• concentration: typical concentration used in the crystallization is 10 mg/ml (0.5 mM for 20 kDa protein) but this may vary (2-50 mg/ml). Protein should be soluble and solubility usually determines the used concentration

• amount of protein: 1 mg of protein is needed for initial crystallization screens but typically 10 mg is needed for new crystal structure determination

• temperature: freezing should be avoided, protein is handled gently and stored at 4 C.

Steps in the crystallization:

• screening: initial crystallization conditions are typically searched by using random or grid screens. Typical single screen contains 50 different conditions. Proteins are crystallized by using salts, polyethylene glycol, and small organic compounds such as detergents. pH is an important parameter.

• crystal growth optimization: crystal quality and size is optimized by varying crystallization conditions. Typical crystal size varies from 10 micrometer to 1 millimeter

• diffraction tests: in-house X-ray diffractometer is used to test if the obtained crystals are protein or salt crystasl as well as diffraction-quality

STRUCTURE DETERMINATION

Steps in the structure determination:

• data collection: diffraction data sets are collected by using in-house diffractometer. High-resolution data sets are collected by using synchrotron radiation.

• structure solution: for example, heavy atom derivatives or molecular replacement are used for solving the phase problem. This allows the calculation of electron density maps. Protein model is built by using appropriate software and checked by using molecular graphics

• the result is the set of coordinates for atoms in the PDB (Protein Data Bank) format

STRUCTURE INTERPRETATION

The crystal structure of a protein is rich in information. The three-dimensional structures can be used for the interpretation of the function and properties of protein. The protein crystal structures are widely used, for example, in protein engineering and drug design.