Structure determination of an integral membrane kinase

Cell-Free Expression and in Meso Crystallisation of an Integral Membrane Kinase for Structure Determination.

DIACYLGLYCEROL KINASE - DELTA 7, DAGK, DIGLYCERIDE KINASE, DGK

3-5 %(V/V) 2-METHYL-2, 4-PENTANEDIOL (MPD), 0.1 M SODIUM CHLORIDE, 0.06 M MAGNESIUM ACETATE, 0.1 M SODIUM CITRATE/HCL PH 5.6. CRYSTALLIZED USING THE IN MESO (LIPIDIC CUBIC PHASE, LCP) METHOD AT 4 DEGREES CELCIUS WITH THE 7.8 MONOACYLGLYCEROL (7.8 MAG) AS THE HOSTING LIPID.

THE CONSTRUCT CONTAINS AN N-TERMIANL HIS TAG 'GHHHHHHEL'. COMPARED TO THE WILDTYPE FORM, THE PROTEIN HAS SEVEN MUTATIONS. THEY ARE A41C, C46A, I53V, I70L, M96L, V107D AND C113A.

THERE ARE SIX NCS-RELATED MOLECULES IN THE ASYMMETRIC UNIT BUT NCS RESTRAINTS WERE NOT USED IN THE REFINEMENT.

TRANSFERASE, CELL-FREE, DGKA, IN MESO CRYSTALLIZATION, IN VITRO EXPRESSION, LIPID METABOLISM, LIPIDIC MESOPHASE, LIPIDIC CUBIC PHASE, LCP, MEMBRANE PROTEIN, MICROCRYSTAL, 7.8 MAG, THERMOSTABLE MUTANT