Structure determination protein crystallography

In the early days of protein crystallography the determination of a protein structure was laborious and time consuming. The diffracted beams were obtained from weak x-ray sources and recorded on films that had to be manually scanned and measured. The available computers were far from adequate for the problem, with a computing power roughly equal to present-day pocket calculators. Computer graphics were not available, and models of the protein had to be built manually from pieces of steel rod. To determine the... 

The three-dimensional structure of protein molecules can be experimentally determined by two different methods, x-ray crystallography and NMR. The interaction of x-rays with electrons in molecules arranged in a crystal is used to obtain an electron-density map of the molecule, which can be interpreted in terms of an atomic model. Recent technical advances, such as powerful computers including graphics work stations, electronic area detectors, and... 

More detailed aspects of protein function can be obtained also by force-field based approaches. Whereas protein function requires protein dynamics, no experimental technique can observe it directly on an atomic scale, and motions have to be simulated by molecular dynamics (MD) simulations. Also free energy differences (e.g. between binding energies of different protein ligands) can be characterised by MD simulations. Molecular mechanics or molecular dynamics based approaches are also necessary for homology modelling and for structure refinement in X-ray crystallography and NMR structure determination. 

This chapter consists of four main sections. The first provides an overall description of the process of contemporary protein structure determination by X-ray crystallography and summarizes the current computational requirements. This is followed by a summary and examples of the use of structure-based methods in drug discovery. The third section reviews the key developments in computer hardware and computational methods that have supported the development and application of X-ray crystallography over the past forty or so years. The final section outlines the areas in which improved... 

For many proteins, it is possible to generate structures of protein-ligand complexes quite rapidly. It is therefore not uncommon for many hundreds of structures to be determined in support of a drug discovery and optimization project. The major challenge for this level of throughput is informatics support. It is also this type of crystallography that is most in need of semiautomated procedures for structure solution and model building (see Section 12.6). 

There has been considerable and continuing investment in e-science and Grid-based computing around the world. Of particular interest for protein crystallography is the e-HTPX project funded by the UK research councils (http //www.e-htpx.ac.uk). The aim of e-HTPX is to unify the procedures of protein structure determination into a single all-encompassing interface from which users can initiate, plan, direct, and document their experiment either locally or remotely from a desktop computer. 

Figure 5 Steps in the use of protein crystallography for structure determination.

Power and Limits of the SAXS Methods. This field of SAXS is in competition with the field of protein crystallography. The spatial resolution of the SAXS method is limited (> 0.5 nm), whereas structures determined by protein crystallography are exact up to fractions of Angstrpms. On the other hand, the protein crystallography is unable to study living proteins under almost physiological conditions. Moreover, kinetic processes can be monitored by SAXS but cannot be studied by means of protein crystallography. 

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. 

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