0.95 mM [U-100% 13C; U-100% 15N] protein, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 20 mM MES, 0.02 % by volume NaN3, 50 uM DSS, 90% H2O/10% D2O
90% H2O/10% D2O
2
0.975 mM [U-5% 13C; U-100% 15N] protein, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 20 mM MES, 0.02 % by volume NaN3, 50 uM DSS, 90% H2O/10% D2O
90% H2O/10% D2O
3
0.664 mM [U-5% 13C; U-100% 15N] protein, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 20 mM MES, 0.02 % by volume NaN3, 50 uM DSS, 4 % by volume poly ethylene glycol, 82% H2O/18% D2O
82% H2O/18% D2O
4
0.851 mM [U-5% 13C; U-100% 15N] protein, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 20 mM MES, 0.02 % by volume NaN3, 50 uM DSS, 7 % by volume poly acrylamide gel, 83% H2O/17% D2O
83% H2O/17% D2O
試料
濃度 (mg/ml)
構成要素
Isotopic labeling
Solution-ID
0.95mM
protein-1
[U-100% 13C; U-100% 15N]
1
100mM
sodium chloride-2
1
5mM
calcium chloride-3
1
10mM
DTT-4
1
20mM
MES-5
1
0.02 %
NaN3-6
1
50uM
DSS-7
1
0.975mM
protein-8
[U-5% 13C; U-100% 15N]
2
100mM
sodium chloride-9
2
5mM
calcium chloride-10
2
10mM
DTT-11
2
20mM
MES-12
2
0.02 %
NaN3-13
2
50uM
DSS-14
2
0.664mM
protein-15
[U-5% 13C; U-100% 15N]
3
100mM
sodium chloride-16
3
5mM
calcium chloride-17
3
10mM
DTT-18
3
20mM
MES-19
3
0.02 %
NaN3-20
3
50uM
DSS-21
3
4 %
poly ethylene glycol-22
3
0.851mM
protein-23
[U-5% 13C; U-100% 15N]
4
100mM
sodium chloride-24
4
5mM
calcium chloride-25
4
10mM
DTT-26
4
20mM
MES-27
4
0.02 %
NaN3-28
4
50uM
DSS-29
4
7 %
poly acrylamide gel-30
4
試料状態
イオン強度: 117.5 / pH: 6.5 / 圧: ambient / 温度: 298 K
-
NMR測定
NMRスペクトロメーター
タイプ
製造業者
モデル
磁場強度 (MHz)
Spectrometer-ID
Varian INOVA
Varian
INOVA
750
1
Varian INOVA
Varian
INOVA
600
2
Bruker Avance
Bruker
AVANCE
900
3
-
解析
NMR software
名称
バージョン
開発者
分類
VnmrJ
2.1B
Varian
collection
NMRPipe
2007.030.16.06
Delaglio, Grzesiek, Vuister, Zhu, PfeiferandBax
解析
PROSA
6.4
Guntert
解析
XEASY
Bartelsetal.
データ解析
AutoAssign
Zimmerman, Moseley, KulikowskiandMontelione
chemicalshiftassignment
CYANA
3
Guntert, MumenthalerandWuthrich
構造決定
AutoStructure
2.2.1
Huang, Tejero, PowersandMontelione
構造決定
TALOS+
1.2009.0721.18
Shen, Cornilescu, DelaglioandBax
データ解析
CARA
1.8.4
KellerandWuthrich
chemicalshiftassignment
CARA
1.8.4
KellerandWuthrich
peakpicking
CARA
1.8.4
KellerandWuthrich
データ解析
CNS
1.2.1
Brunger, Adams, Clore, Gros, NilgesandRead
精密化
PSVS
1.3
BhattacharyaandMontelione
構造検証
NMRDraw
3
Delaglio, Grzesiek, Vuister, Zhu, PfeiferandBax
データ解析
MOLMOL
Koradi, BilleterandWuthrich
精密化
TopSpin
2.1
BrukerBiospin
データ解析
精密化
手法: simulated annealing / ソフトェア番号: 1 詳細: Structure determination was performed iteratively with CYANA v3.0 using NOE-based constraints, PHI and PSI dihedral angle constraints from TALOS+, and RDC constraints. The 20 conformers out ...詳細: Structure determination was performed iteratively with CYANA v3.0 using NOE-based constraints, PHI and PSI dihedral angle constraints from TALOS+, and RDC constraints. The 20 conformers out of 100 with the lowest target function were further refined by simulated annealing in explicit water bath using the program CNS with PARAM19 force field.
NMR constraints
NOE constraints total: 3271 / NOE intraresidue total count: 525 / NOE long range total count: 782 / NOE medium range total count: 1120 / NOE sequential total count: 844
代表構造
選択基準: lowest energy
NMRアンサンブル
コンフォーマー選択の基準: target function / 計算したコンフォーマーの数: 100 / 登録したコンフォーマーの数: 14