automated-protocols-for-macromolecular-crystallization-at-mrc

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

Medical Research Council

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.

The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

The Pi sampling method is derived from the incomplete factorial approach to macromolecular crystallization screen design. The resulting `Pi screens' have a modular distribution of a given set of up to 36 stock solutions. Maximally diverse conditions can be produced by taking into account the properties of the chemicals used in the formulation and the concentrations of the corresponding solutions. The Pi sampling method has been implemented in a web-based application that generates screen formulations and recipes. It is particularly adapted to screens consisting of 96 different conditions. The flexibility and efficiency of Pi sampling is demonstrated by the crystallization of soluble proteins and of an integral membrane-protein sample.

Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening.

Progress in macromolecular crystallography depends on further miniaturization of crystallization experiments.

In order to solve increasingly challenging protein structures with crystallography, crystallization reagents and screen formulations are regularly investigated. Here, we briefly describe 96-condition screens developed at the MRC Laboratory of Molecular Biology: the LMB sparse matrix screen, Pi incomplete factorial screens, the MORPHEUS grid screens and the ANGSTROM optimization screen. In this short review, we also discuss the difficulties and advantages associated with the development of protein crystallization screens.

Article	Year
The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors. Nature chemical biology\| PubMed ID: 20081827	2010
Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening. Acta crystallographica. Section D, Biological crystallography\| PubMed ID: 21543849	2011
Progress in macromolecular crystallography depends on further miniaturization of crystallization experiments. Drug discovery today\| PubMed ID: 25048082	2014
Protein crystallization screens developed at the MRC Laboratory of Molecular Biology. Drug discovery today\| PubMed ID: 27032894	2016

Fabrice Gorrec

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