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Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Original publication

DOI

10.1107/s0907444905007808

Type

Journal article

Journal

Acta crystallographica. Section D, Biological crystallography

Publication Date

06/2005

Volume

61

Pages

651 - 657

Addresses

Oxford Protein Production Facility, Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, Oxford OX3 7BN, England.

Keywords

Animals, Humans, Proteins, Crystallography, X-Ray, Nanotechnology, Automation