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

Koruza, Katarina LU ; Lafumat, Bénédicte LU ; Nyblom, Maria ; Knecht, Wolfgang LU and Fisher, Zoë LU (2018) In Crystals 8(11).
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... (More)

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.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Crystallization, Human carbonic anhydrase ix, Microseed matrix screening, Neutron protein crystallography, Optimization, Seeding
in
Crystals
volume
8
issue
11
article number
434
publisher
MDPI AG
external identifiers
  • scopus:85057286372
ISSN
2073-4352
DOI
10.3390/cryst8110434
language
English
LU publication?
yes
id
34dda086-b2d1-43f9-8ccd-489babb4c2bc
date added to LUP
2018-12-04 08:57:43
date last changed
2022-03-09 22:56:45
@article{34dda086-b2d1-43f9-8ccd-489babb4c2bc,
  abstract     = {{<p>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.</p>}},
  author       = {{Koruza, Katarina and Lafumat, Bénédicte and Nyblom, Maria and Knecht, Wolfgang and Fisher, Zoë}},
  issn         = {{2073-4352}},
  keywords     = {{Crystallization; Human carbonic anhydrase ix; Microseed matrix screening; Neutron protein crystallography; Optimization; Seeding}},
  language     = {{eng}},
  month        = {{11}},
  number       = {{11}},
  publisher    = {{MDPI AG}},
  series       = {{Crystals}},
  title        = {{From initial hit to crystal optimization with microseeding of human carbonic anhydrase IX—A case study for neutron protein crystallography}},
  url          = {{http://dx.doi.org/10.3390/cryst8110434}},
  doi          = {{10.3390/cryst8110434}},
  volume       = {{8}},
  year         = {{2018}},
}