| 1. |
- Kelpšas, Vinardas, et al.
(författare)
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Perdeuteration, large crystal growth and neutron data collection of Leishmania mexicana triose-phosphate isomerase E65Q variant
- 2019
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Ingår i: Acta crystallographica. Section F, Structural biology communications. - 2053-230X. ; 75:4, s. 260-269
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Tidskriftsartikel (refereegranskat)abstract
- Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.
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| 2. |
- Koruza, Katarina, et al.
(författare)
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From initial hit to crystal optimization with microseeding of human carbonic anhydrase IX—A case study for neutron protein crystallography
- 2018
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Ingår i: Crystals. - : MDPI AG. - 2073-4352. ; 8:11
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Tidskriftsartikel (refereegranskat)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.
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