From initial hit to crystal optimization with microseeding of human carbonic anhydrase IX—A case study for neutron protein crystallography

Research output: Contribution to journalArticle

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.

Details

Authors
Organisations
External organisations
  • European Spallation Source ESS AB
  • Lund University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Structural Biology

Keywords

  • Crystallization, Human carbonic anhydrase ix, Microseed matrix screening, Neutron protein crystallography, Optimization, Seeding
Original languageEnglish
Article number434
JournalCrystals
Volume8
Issue number11
Publication statusPublished - 2018 Nov 9
Publication categoryResearch
Peer-reviewedYes

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