Structural Analysis of Mutant GltPh Transporter
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Other organizations : Cornell University
Protocol cited in 14 other protocols
Variable analysis
- K55C/C321A/A364C mutation introduced within a heptahistidine mutant of Glt_Ph
- Crystallization and structural determination of the mutant protein
- Purified protein cross-linked in the presence of 10 fold molar excess of HgCl2
- Purified protein dialyzed against buffer containing 10 mM HEPES/NaOH, 7 mM n-decyl-β-D-maltopyranoside, 100 mM NaCl and 100 μM L-asp
- Purified protein diluted to the final concentration of 2–4 mg/ml and supplemented with 0.5 mM E. coli total polar lipid extract and 100 mM NaBr
- Protein solution mixed at 1:1 (v:v) ratio with the reservoir solution, containing 100 mM MES, pH 5.0, 18–20 % PEG 350 MME and 200 mM CaCl2
- Crystallization performed at 4 °C by hanging drop vapour diffusion
- Crystals cryoprotected by allowing the drop to dry until its volume was reduced by 50 %
- Selenomethionine-substituted protein expressed and crystallized as above
- None mentioned
- None mentioned
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