X-ray crystallography requirements

Optimally, X-ray crystallography requires single crystals of a few tenths of a millimetre in size, which are not intergrown and which have grown slowly (they are usually of better quality). Neutron experiments generally require larger crystals a decade ago ca. 5mm was considered a standard size, but recent improvement of the technique make considerably smaller samples acceptable (<1 mm ). 

Both NMR and x-ray crystallography require large investments in analytical instruments and computers to analyze the data, entailing multimillion dollar investments just to analyze the structure of potential drug receptors. The justification for such investments, made by many firms in recent years, is that defining the three-dimensional structure of receptor sites will improve the prospects for developing drugs that fit into those sites. The wisdom of the investments will only become clear in several years, if this approach bears fruit. 

As will be evident from the foregoing paragraph, this ingenious experiment was a rather difficult problem to carry out in practice, designing (or imagining) the appropriate compounds, the synthesis (or acquisition) of all of those compounds, and then the X-ray crystallography required to provide the information. And how did this come out It came out very well indeed. And we will outhne this work here in some detail. 

Physical, chemical, and biological properties are related to the 3D structure of a molecule. In essence, the experimental sources of 3D structure information are X-ray crystallography, electron diffraction, or NMR spectroscopy. For compounds without experimental data on their 3D structure, automatic methods for the conversion of the connectivity information into a 3D model are required (see Section 2.9 of this Textbook and Part 2, Chapter 7.1 of the Handbook) [16]. 

To obtain the secondary and tertiary stmcture, which requires detailed information about the arrangement of atoms within a protein, the main method so far has been x-ray crystallography. In recent years NMR methods have been developed to obtain three-dimensional models of small protein molecules, and NMR is becoming increasingly useful as it is further developed. 

Although great progress has been made in study of the mode of action of the azinomycins, a full understanding of their interaction with DNA will require future structural characterization of azinomycin-DNA crosslinks by NMR or X-ray crystallography. 

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... 

In 1958 Barker (20) isolated a red, heat stable, light labile, cofactor which was required for the metabolism of glutamate in cell-extracts of Clostridium tetanomorphum. Subsequently this cofactor was crystallized. X-ray crystallography identified Barker s cofactor as the coenzyme form of Vitamin B12 (15, 21). 

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. 

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