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Crystals 2018, 8(12), 464; https://doi.org/10.3390/cryst8120464

A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements

1
Office of Educational Programs, Brookhaven National Laboratory, Upton, NY 11973, USA
2
Department of Pharmacology, Stony Brook University, Stony Brook, NY 11794, USA
3
School of Business, State University of New York at Old Westbury, Old Westbury, NY 11568, USA
4
Department of Biology, College of William and Mary, Williamsburg, VA 23187, USA
5
General Douglas MacArthur High School, Levittown, NY 11756, USA
6
Ross School, East Hampton, NY 11937, USA
7
Shoreham-Wading River High School, Shoreham-Wading River Central School District, Shoreham, NY 11786, USA
8
Photon Sciences Directorate, Brookhaven National Laboratory, Upton, NY 11973, USA
9
Biosciences Department, Brookhaven National Laboratory, Upton, NY 11973, USA
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 14 November 2018 / Revised: 7 December 2018 / Accepted: 10 December 2018 / Published: 12 December 2018
(This article belongs to the Special Issue Biological Crystallization)
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Abstract

A technique is described for generating large well diffracting crystals from conditions that yield microcrystals. Crystallization using this technique is both rapid (crystals appear in <1 h) and robust (48 out of 48 co-crystallized with a fragment library, compared with 26 out of 48 using conventional hanging drop). Agarose gel is used to exclude nucleation inducing elements from the remaining crystallization cocktail. The chemicals in the crystallization cocktail are partitioned into high concentration components (presumed to induce aggregation by reducing water activity) and low concentration nucleation agents (presumed to induce nucleation through direct interaction). The nucleation agents are then combined with 2% agarose gel and deposited on the crystallization shelf of a conventional vapor diffusion plate. The remaining components are mixed with the protein and placed in contact with the agarose drop. This technique yielded well diffracting crystals of lysozyme, cubic insulin, proteinase k, and ferritin (ferritin crystals diffracted to 1.43 Å). The crystals grew rapidly, reaching large size in less than one hour (maximum size was achieved in 1–12 h). This technique is not suitable for poorly expressing proteins because small protein volumes diffuse out of the agarose gel too quickly. However, it is a useful technique for situations where crystals must grow rapidly (such as educational applications and preparation of beamline test specimens) and in situations where crystals must grow robustly (such as co-crystallization with a fragment library). View Full-Text
Keywords: drug discovery; education; crystallization; crystallography; nucleation; micro-crystals; agarose; ferritin; lysozyme; proteinase k; insulin drug discovery; education; crystallization; crystallography; nucleation; micro-crystals; agarose; ferritin; lysozyme; proteinase k; insulin
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Blackburn, A.; Partowmah, S.H.; Brennan, H.M.; Mestizo, K.E.; Stivala, C.D.; Petreczky, J.; Perez, A.; Horn, A.; McSweeney, S.; Soares, A.S. A Simple Technique to Improve Microcrystals Using Gel Exclusion of Nucleation Inducing Elements. Crystals 2018, 8, 464.

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