温度: 291 K / 手法: 蒸気拡散法, ハンギングドロップ法 / pH: 8.5 詳細: PROTEINS WERE DIALYZED IN A BUFFER OF 10 MM TRIS-HCL, PH 8.5, 1 MM DTT. THE PROTEIN WAS CONCENTRATED TO 10 MG/ML USING AMICON ULTRAFREE CENTRIFUGAL FILTER DEVICES, AND INHIBITOR, AC-DEVD-CMK ...詳細: PROTEINS WERE DIALYZED IN A BUFFER OF 10 MM TRIS-HCL, PH 8.5, 1 MM DTT. THE PROTEIN WAS CONCENTRATED TO 10 MG/ML USING AMICON ULTRAFREE CENTRIFUGAL FILTER DEVICES, AND INHIBITOR, AC-DEVD-CMK RECONSTITUTED IN DMSO, WAS THEN ADDED AT 5:1 WT:WT, INHIBITOR TO PEPTIDE. THE PROTEIN WAS DILUTED TO A CONCENTRATION OF 8 MG/ML BY ADDING 10 MM TRIS-HCL, PH 8.5, CONCENTRATED DTT AND CONCENTRATED NAN3 SO THAT THE FINAL BUFFER WAS 10 MM TRIS-HCL, PH 8.5, 10 MM DTT, 3 MM NAN3. 2 UL OF CONCENTRATED PROTEIN WAS MIXED 1:1 WITH WELL BUFFER THAT CONTAINED 100 MM SODIUM CITRATE, PH 5, 3 MM NAN3, 10 MM DTT AND 17% PEG 6000 W/V. SOLUTIONS WERE INCUBATED AT 18 DEG C USING THE HANGING DROP METHOD. CRYSTALS GREW WITHIN THREE DAYS FOR WILD- TYPE CASPASE-3 AND WITHIN TWO WEEKS FOR THE MUTANTS. THE IDEAL FREEZING CONDITIONS WERE FOUND TO BE WITH 80% MOTHER LIQUOR AND 20% PEG 400.
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データ収集
回折
平均測定温度: 100 K
放射光源
由来: シンクロトロン / サイト: APS / ビームライン: 22-ID / 波長: 1
検出器
タイプ: MARRESEARCH / 検出器: CCD
放射
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
波長: 1 Å / 相対比: 1
反射
解像度: 1.4→50 Å / Num. obs: 54279 / % possible obs: 97.9 % / Observed criterion σ(I): 2 / 冗長度: 5.8 % / Biso Wilson estimate: 21.1 Å2 / Rmerge(I) obs: 0.06 / Net I/σ(I): 46.2
反射 シェル
解像度: 1.4→1.45 Å / 冗長度: 3.5 % / Rmerge(I) obs: 0.75 / Mean I/σ(I) obs: 1.35 / % possible all: 83.8
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解析
ソフトウェア
名称
バージョン
分類
CNS
1.1
精密化
HKL-2000
データ削減
SCALEPACK
データスケーリング
精密化
構造決定の手法: 分子置換 / 解像度: 1.4→27.01 Å / Rfactor Rfree error: 0.003 / Data cutoff high absF: 1033475.18 / Isotropic thermal model: RESTRAINED / 交差検証法: THROUGHOUT / σ(F): 0