The native MetNIQ and selenomethionine-substituted MetNIQ complexes were prepared as described previously (17 (link)). Crystallization screens for MetNIQ were conducted at different concentrations, 5–20 mg/mL, by vapor diffusion in hanging drops at 20 °C using three different commercial kits, including MemGold2, MORPHEUS (Molecular Dimension), and PEGRx (Hampton Research). MetNIQ was crystallized in a reservoir containing 0.1 M Mes, pH 6, and 22% PEG 400, using 5 mg/mL protein at a ratio of protein to reservoir of 2:1. Crystals appeared after 2–3 d, fully grew after 5–7 d, and shattered after 10–14 d. Crystals were harvested at day 7 and cryoprotected by increasing PEG 400 concentration to 25, 30, and then 35%, followed by flash-freezing for data collection. Selenomethionine-substituted MetNIQ crystals often diffracted better than the native ones.
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Crystallization of MetNIQ Protein Complexes
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Corresponding Organization : Howard Hughes Medical Institute
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Variable analysis
independent variables
- Protein concentration (5-20 mg/mL)
- Crystallization conditions (0.1 M Mes, pH 6, and 22% PEG 400)
- Crystallization success (whether crystals formed)
- Diffraction quality of crystals
- Temperature (20 °C)
- Vapor diffusion method
- Commercial crystallization kits used (MemGold2, MORPHEUS, PEGRx)
- Native MetNIQ complex
- Selenomethionine-substituted MetNIQ complex
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