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A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization

Open access
Walter, Thomas S., Diprose, Jonathan M., Mayo, Chris J., Siebold, Christian, Pickford, Mike G., Carter, Lester, Sutton, Geoff C., Berrow, Nick S., Brown, James, Berry, Ian M., Stewart-Jones, Guillaume B. E., Grimes, Jonathan M., Stammers, David K., Esnouf, Robert M., Jones, E. Yvonne, Owens, Ray J., Stuart, David I., Harlos, Karl·2005·Acta Crystallographica Section D Biological Crystallography

Abstract

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.

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Metadata

Authors
Walter, T. S., Diprose, J. M., Mayo, C. J., Siebold, C., Pickford, M. G., Carter, L., Sutton, G. C., Berrow, N. S., Brown, J., Berry, I. M., Stewart-Jones, G. B. E., Grimes, J. M., Stammers, D. K., Esnouf, R. M., Jones, E. Y., Owens, R. J., Stuart, D. I., & Harlos, K.
Venue
Acta Crystallographica Section D Biological Crystallography
Published
2005-05-26
Volume
61
Issue
6
Pages
651-657
ISSN
0907-4449 (ISSN-L)
OpenAlex
W1993442892
Cited by
247
References
0
FWCI
10.48
Type
journal article
Language
en
Open access
hybrid

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CoChat. (2026). Citation check: A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. https://app.cochat.ai/papers/10.1107/s0907444905007808
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Walter, T. S., Diprose, J. M., Mayo, C. J., Siebold, C., Pickford, M. G., Carter, L., Sutton, G. C., Berrow, N. S., Brown, J., Berry, I. M., Stewart-Jones, G. B. E., Grimes, J. M., Stammers, D. K., Esnouf, R. M., Jones, E. Y., Owens, R. J., Stuart, D. I., & Harlos, K. (2005). A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallographica Section D Biological Crystallography. https://doi.org/10.1107/s0907444905007808

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