Protein crystallography crystallization

The potential application of self-assembled scaffolds offering exact positioning of biological macromolecules within micron- to milimeter-size DNA- or protein-made crystals for structural analysis by x-ray crystallography was also suggested [5,50]. 

Obtaining large single crystals that diffract to high resolution remains the primary bottleneck of protein crystallography. The most widely used... 

Protein crystallography often requires special constructs or mutants to facilitate crystallization it also requires large quantities of highly purified protein. Thus to move forward in a timely fashion, it is important that an industrial structural biology group employ molecular biologists and individuals with expertise in protein purification. 

Protein-crystallography has revealed many of these groups as binding centers of heavy metals such as Pt(II), Hg(II), Au(III). It is important to note that the solution conditions in which the reagent has been applied to these crystals differ widely. If an acid pH has been used then groups with a high affinity for protons. 

Until quite recently, X-ray crystallography was the technique used almost exclusively to resolve the three-dimensional structure of proteins. As well as itself being technically challenging, a major limitation of X-ray crystallography is the requirement for the target protein to be in crystalline form. It has thus far proven difficult/impossible to induce the majority of proteins to crystallize. NMR is an analytical technique that can also be used to determine the three-dimensional structure of a molecule, and without the necessity for crystallization. For many years, even the most powerful NMR machines could resolve the three-dimensional structure of only relatively small proteins (less than 20-25 kDa). However, recent analytical advances now render it possible to analyse much larger proteins by this technique successfully. 

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

Homologous proteins. Homologous proteins usually crystallize under very different conditions. For example, in our laboratory two homologous proteins crystallize in either 10 mM sodium acetate, pH 3.8, 5 mM DTT, and 50% (v/v) MPD (Weichsel et al, 1996), or 100 mM ammonium sulfate, 30% PEG 6000, and 10 mM DTT (Filson et al, 2003). This technique was first used by Kendrew for solving the structure of sperm whale myoglobin (Kendrew et al., 1954) and has been used in many other structural studies. Currently, this technique is heavily exploited in membrane protein crystallography (Wiener,... 

For protein crystallography, the repository of most protein crystal structures is the PDB hosted at http // www.rcsb.org/pdb/ (Berman et al., 2000). This database contains the 3-D coordinates (and sometimes the structure factor files) for almost all protein crystal structures. Most journals currently require deposition of the coordinates when pubhshing stmcture papers. Each structure is given a unique identification code that will be listed in the paper (see Figure 22-1 for examples of PDB codes). Structures can be accessed using this code, or using various other search criteria. The PDB also contains structural information for NMR structures. 

A fully automated protein crystallography beamline at a third-generation synchrotron source can screen several hundred crystals daily. Automatic evaluation of the diffraction images to ascertain crystal quality is, therefore, a critical step for high-throughput data acquisition. Evaluation of each image requires software that mimics the traditional visual assessment of crystal quality. 

Muchmore, S. W., Olson, J., Jones, R., Pan, J., Blum, M., Greer, J., Merrick, S. M., Magdalinos, P and Nienaber, V. L. (2000). Automated crystal mounting and data collection for protein crystallography. Structure 8, R243-R246. 

Muchmore, S. W.,etal. (2000). Automated crystal mounting and data collection for protein crystallography. Structure Fold Des. 8, R243-246. 

Jin, L. and Babine, R. E. (2004). Engineering proteins to promote crystallization. In Protein Crystallography in Drug Discovery. Methods and Principles in Medicinal Chemistry, Babine, R. E. and Abdel-Meguid, S. S., eds, Vol. 20, Wiley-VCH, pp. 209-216. 

The problem of phase determination is the fundamental one in any crystal structure analysis. Classically protein crystallography has depended on the method of multiple isomorphous replacement (MIR) in structure determination. However lack of strict isomorphism between the native and derivative crystals and the existence of multiple or disordered sites limit the resolution to which useful phases may be calculated. 

All the above techniques use incident monochromatic radiation, usually focus in one or two dimensions. However for cases a) and d) the reduction of radiation damage and more particularly in kinetic crystallography the use of polychromatic data collection is yielding promising results. This technique makes combined use of the intensity and collimation of the SR beam with a large wavelength spread for Laue data colla tion from protein single crystals. 

Crystal structure of a protein molecule can also be determined by x-ray crystallography. Purified protein is crystallized either by batch methods or vapor diffusion. X-rays are directed at a crystal of protein. The rays are scattered depending on the electron densities in different positions of a protein. Images are translated onto electron density maps and then analyzed computationally to construct a model of the protein. It is especially important for structure-based drug designs. 

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