Crystal structure of the integral membrane diacylglycerol kinase - delta7

Crystal Structure of the Integral Membrane Diacylglycerol Kinase.

THE WILD-TYPE GENE WAS SYNTHESIZED BASED ON THE DGKA NUCLEOTIDE SEQUENCE OF ESCHERICHIA COLI K12, WITH ADDITIONAL NUCLEOTIDES ENCODING HIS TAG SEQUENCES AT THE N- TERMINUS. SITE-DIRECTED MUTATIONS WERE MADE USING PCR.

3-5 %(V/V) 2-METHYL-2, 4-PENTANEDIOL, 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) METHOD AT 4 DEGREE CELSIUS WITH THE 7.8 MONOACYLGLYCEROL (7.8 MAG) AS THE HOSTING LIPID.

CRYSTAL STRUCTURE OF THE INTEGRAL MEMBRANE DIACYLGLYCEROL KINASE - WILD-TYPE

CRYSTAL STRUCTURE OF THE INTEGRAL MEMBRANE DIACYLGLYCEROL KINASE - DELTA4

(2S)-2,3-DIHYDROXYPROPYL(7Z)-PENTADEC-7-ENOATE (78M): ALSO CONTAINS THE R-ISOMER OF THIS 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.

THE 6 CHAINS FORMING 2 TRIMERS PER ASYMMETRIC UNIT COULD BE RELATED BY NCS HOWEVER THIS WAS DETRIMENTAL TO REFINEMENT AND SO WAS NOT USED. FOR THE MAJORITY OF THE REFINEMENT THE PHASES FROM SEMET DATA WERE USED, TARGET MLHL, UNTIL THE FINAL ROUNDS OF REFINEMENT WHERE TARGET WAS ML. THE FOLLOWING RESIDUES COULD NOT BE PLACED INTO ELECTRON DENSITY DUE TO DISORDER OR FLEXIBILITY - A1-5, B1-23, C1-31, D1-13, E1-35, E47-49, F1-33.

TRANSFERASE, LIPID METABOLISM, IN MESO CRYSTALLISATION, LIPID CUBIC PHASE, LIPIDIC MESOPHASE, THERMOSTABLE MUTANT, MONOACYLGLYCEROL, 7.8 MAG