分子量: 14204.451 Da / 分子数: 6 / 変異: YES / 由来タイプ: 組換発現 / 由来: (組換発現) ESCHERICHIA COLI K-12 (大腸菌) 解説: 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 ...解説: 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. プラスミド: PTRCHISB_DGKA_DELTA7 / 発現宿主: ESCHERICHIA COLI (大腸菌) / 株 (発現宿主): WH1061 / 参照: UniProt: P0ABN1, diacylglycerol kinase (ATP)
(2S)-2,3-DIHYDROXYPROPYL(7Z)-PENTADEC-7-ENOATE (78M): ALSO CONTAINS THE R-ISOMER OF THIS LIPID
配列の詳細
THE CONSTRUCT CONTAINS AN N-TERMIANL HIS TAG 'GHHHHHHEL'. COMPARED TO THE WILDTYPE FORM, THE ...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.
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実験情報
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実験
実験
手法: X線回折 / 使用した結晶の数: 19
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試料調製
結晶
マシュー密度: 3.95 Å3/Da / 溶媒含有率: 68.86 % / 解説: NONE
結晶化
温度: 277 K / 手法: 脂質キュービック相法 / pH: 5.6 詳細: 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 ...詳細: 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.
モノクロメーター: SI(111) DOUBLE CRYSTAL CRYO-COOLED プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: x-ray
放射波長
ID
波長 (Å)
相対比
1
1.0332
1
2
0.97944
1
反射
解像度: 2.05→58.04 Å / Num. obs: 62716 / % possible obs: 99.7 % / Observed criterion σ(I): -3 / 冗長度: 9.3 % / Biso Wilson estimate: 42.5 Å2 / Rmerge(I) obs: 0.11 / Net I/σ(I): 12.4
反射 シェル
解像度: 2.05→2.16 Å / 冗長度: 6.9 % / Rmerge(I) obs: 0.85 / Mean I/σ(I) obs: 2 / % possible all: 99.5
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解析
ソフトウェア
名称
バージョン
分類
PHENIX
(PHENIX.REFINE)
精密化
XDS
データ削減
XSCALE
データスケーリング
SHELX
CDE
位相決定
精密化
構造決定の手法: 単波長異常分散 開始モデル: NONE 解像度: 2.05→51.894 Å / SU ML: 0.23 / σ(F): 0 / 位相誤差: 20.52 / 立体化学のターゲット値: ML 詳細: 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 ...詳細: 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.
Rfactor
反射数
%反射
Rfree
0.2167
3161
5.1 %
Rwork
0.1959
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obs
0.1969
62622
99.66 %
溶媒の処理
減衰半径: 0.9 Å / VDWプローブ半径: 1.11 Å / 溶媒モデル: FLAT BULK SOLVENT MODEL