Proteins crystallography

Computer Applications in Pharmaceutical Research and Development, Edited by Sean Ekins. ISBN 0-471-73779-8 Copyright 2006 John Wiley Sons, Inc. 

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 

A major requirement for structural studies is the availability of large quantities of pure, functional protein. Essentially all proteins are present in only very small quantities in native cells or tissues, so molecular biology techniques 

Final structure Fit eiectron density Solve structure Collect diffraction data 

A regularly formed crystal of reasonable size (typically 500 pm in each dimension) is required for X-ray diffraction. Samples of pure protein are screened against a matrix of buffers, additives, or precipitants for conditions under which they form crystals. This can require many thousands of trials and has benefited from increased automation over the past five years. Most large crystallographic laboratories now have robotics systems, and the most sophisticated also automate the visualization of the crystallization experiments, to monitor the appearance of crystalline material. Such developments [e.g., Ref. 1] are adding computer visualization and pattern recognition to the informatics requirements. 

Amino acids are linked together to form polymers via the reaction of the acidic -COOH group 

For a satisfactory crystallographic study, it is vital to start with a high quality crystal. This is not easy for proteins. Crystallisation of such large molecular complexes frequently leads to trapped solvent, and a reasonable protein crystal may still contain between 30-80% solvent. Because the 

At the outset, crystallographers determine the unit cell dimensions of the protein and its density. The density can yield a value for the molar mass of the protein, provided that the included solvent is taken into account. The space group of the crystal is also important. Protein molecules are enantiomorphous (see Chapter 4), and as a consequence, will only crystallise in space groups with no inversion centres or mirror planes. There are just 65 possibilities from the 230 space groups. 

The major experimental difficulty in protein crystallography is the solution of the phase problem (Section 6.13). There are a number of ways in which this can be tackled the first to be perfected, and the method which is still a cornerstone of protein structural work, is that of isomor-phous replacement. This, and other techniques, is described in the following section. 

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D. E. McRee, Practical Protein Crystallography, Academic Press, Inc., New York, 1993. 

Recent technological advances have greatly influenced 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... 

Eisenberg, D., Hill, C.P. Protein crystallography more surprises ahead. Trends Biochem. Sci. 14 260-264, 1989. 

Lolis, E., and Petsko, G., 1990. Transition-state analogues in protein crystallography Probes of the structural source of enzyme catalysis. Annual Review of Biochemistry 59 597—630. 

The 1980s saw many important developments in the scientific disciplines that underpin the use of protein crystallography in the pharmaceutical and biotechnology industries. Molecular biology and protein chemistry methods... 

The Collaborative Computational Project Number 4 in Protein Crystallography was set up in 1979 to support collaboration between researchers working on such software in the UK and to assemble a comprehensive collection of software to satisfy the computational requirements of the relevant UK groups. The results of this effort gave rise to the CCP4 program suite [45], which is now distributed to academic and commercial users worldwide (see http //www.ccp4.ac.uk). 

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. 

Collaborative computational project, number 4. The CCP4 Suite programs for protein crystallography. Acta Cryst 1994 D50 760-3. 

Babine RE, Abdel-Meguid SS. Protein crystallography in drug discovery (Vol. 20 of Mannhold R, Kubinyi H, Folkers G, editors. Methods and Principles in Medicinal Chemistry). Weinheim Wiley-VCH, 2004. 

The ligands must not include rotatable bonds that cannot be detected by protein crystallography, e.g. hydroxyl torsions. 

Methods that utilize structural data of the target, generally identified by protein crystallography, to look for molecules that complement the binding site through favorable protein-ligand interactions (protein structure-based VS or SBVS). 

Protein Crystallography in Structure-Based Drug Design... 

For most noncrystallographers, protein crystallography tends be a black box full of jargon. Here, we give a brief overview of the technology in an attempt to demystify some of the terms used. 

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

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