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Crystals 2018, 8(11), 434; https://doi.org/10.3390/cryst8110434

From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

1
Department of Biology and Lund Protein Production Platform, Lund University, Sölvegatan 35, 22362 Lund, Sweden
2
Scientific Activities Division, European Spallation Source ERIC, Box 176, 22100 Lund, Sweden
*
Authors to whom correspondence should be addressed.
Received: 22 October 2018 / Revised: 16 November 2018 / Accepted: 17 November 2018 / Published: 20 November 2018
(This article belongs to the Special Issue Biological Crystallization)
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Abstract

Human carbonic anhydrase IX (CA IX) is a multi-domain membrane protein that is therefore difficult to express or crystalize. To prepare crystals that are suitable for neutron studies, we are using only the catalytic domain of CA IX with six surface mutations, named surface variant (SV). The crystallization of CA IX SV, and also partly deuterated CA IX SV, was enabled by the use of microseed matrix screening (MMS). Only three drops with crystals were obtained after initial sparse matrix screening, and these were used as seeds in subsequent crystallization trials. Application of MMS, commercial screens, and refinement resulted in consistent crystallization and diffraction-quality crystals. The crystallization protocols and strategies that resulted in consistent crystallization are presented. These results demonstrate not only the use of MMS in the growth of large single crystals for neutron studies with defined conditions, but also that MMS enabled re-screening to find new conditions and consistent crystallization success. View Full-Text
Keywords: crystallization; microseed matrix screening; seeding; optimization; human carbonic anhydrase IX; neutron protein crystallography crystallization; microseed matrix screening; seeding; optimization; human carbonic anhydrase IX; neutron protein crystallography
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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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Koruza, K.; Lafumat, B.; Nyblom, M.; Knecht, W.; Fisher, Z. From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography. Crystals 2018, 8, 434.

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