Structure of human KCNE1 (also known as MinK) in LMPG micelles. KCNE1 modulates certain voltage- ...Structure of human KCNE1 (also known as MinK) in LMPG micelles. KCNE1 modulates certain voltage-gated potassium channels.
THESE COORDINATES ARE FOR THE R104K MUTANT FORM OF KCNE1, WHICH WAS GENERATED AT AN EARLY STAGE IN ...THESE COORDINATES ARE FOR THE R104K MUTANT FORM OF KCNE1, WHICH WAS GENERATED AT AN EARLY STAGE IN STRUCTURAL DETERMINATION BY A PCR ERROR. THE R104K MUTANT MODULATES KCNQ1 CHANNEL FUNCTION IN WILD TYPE-LIKE MANNER. MUTATION OF THIS ARG RESIDUE TO LYS IS OBSERVED IN MAMMALIAN KCNE1 HOMOLOGS TO THE HUMAN PROTEIN.
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実験情報
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実験
実験
手法: 溶液NMR
NMR実験
Conditions-ID
Experiment-ID
Solution-ID
タイプ
1
1
1
2D TROSY-1H-15N HSQC
1
2
2
3D TROSY-HNCO
1
3
2
3D TROSY-HNCA
1
4
2
3D TROSY-HN(CO)CA
1
5
2
3D TROSY-CBCA(CO)NH
1
6
2
3D TROSY-HN(CA)CB
1
7
3
3D 1H-15N TROSY-NOESY
1
8
4
2D TROSY-1H-15N HSQC
NMR実験の詳細
Text: The structure was determined using combination of NOEs, PREs, and RDCs.
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試料調製
詳細
Solution-ID
内容
溶媒系
1
0.5-1 mM [U-100% 15N] KCNE1 (MinK), 10 % D2O, 2 mM EDTA, 2 mM DTT, 250 mM imidazole, 4 % LMPG, 90% H2O/10% D2O
90% H2O/10% D2O
2
1 mM [U-13C; U-15N; U-2H] KCNE1 (MinK), 10 % D2O, 2 mM EDTA, 2 mM DTT, 250 mM imidazole, 4 % LMPG, 90% H2O/10% D2O
90% H2O/10% D2O
3
1 mM [U-100% 13C; U-100% 15N; 70% 2H] KCNE1 (MinK), 10 % D2O, 2 mM EDTA, 2 mM DTT, 250 mM imidazole, 4 % LMPG, 90% H2O/10% D2O
90% H2O/10% D2O
4
1 mM [U-15N; U-2H] KCNE1 (MinK), 10 % D2O, 2 mM EDTA, 2 mM DTT, 250 mM imidazole, 4 % LMPG, 90% H2O/10% D2O
90% H2O/10% D2O
試料
濃度 (mg/ml)
構成要素
Isotopic labeling
Solution-ID
0.5mM
KCNE1 (MinK)
[U-100% 15N]
1
10 %
D2O
1
2mM
EDTA
1
2mM
DTT
1
250mM
imidazole
1
4 %
LMPG
1
1mM
KCNE1 (MinK)
[U-13C; U-15N; U-2H]
2
10 %
D2O
2
2mM
EDTA
2
2mM
DTT
2
250mM
imidazole
2
4 %
LMPG
2
1mM
KCNE1 (MinK)
[U-100% 13C; U-100% 15N; 70% 2H]
3
10 %
D2O
3
2mM
EDTA
3
2mM
DTT
3
250mM
imidazole
3
4 %
LMPG
3
1mM
KCNE1 (MinK)
[U-15N; U-2H]
4
10 %
D2O
4
2mM
EDTA
4
2mM
DTT
4
250mM
imidazole
4
4 %
LMPG
4
試料状態
イオン強度: 0.25 / pH: 6 / 圧: ambient / 温度: 313 K
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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
Bruker Avance
Bruker
AVANCE
600
1
Bruker Avance
Bruker
AVANCE
800
2
Varian INOVA
Varian
INOVA
900
3
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解析
NMR software
名称
バージョン
開発者
分類
TopSpin
1.3
BrukerBiospin
collection
NMRPipe
v2.4
Delaglio, F. etal.
解析
NMRView
5.2.2_01
Johnson, B.A. etal.
データ解析
CYANA
2.1
Guntert, P. etal.
データ解析
X-PLOR NIH
2.17
Schwieters, C.D. etal.
精密化
ProcheckNMR
v.3.5.4
Laskowski, R.A. etal.
データ解析
精密化
手法: DGSA-distance geometry simulated annealing / ソフトェア番号: 1 詳細: The refinement protocol consisted of slow cooling from 1000 K to 100 K spanning 45 ps. Final structures were energy-minimized using 250 steps of Powell energy minimization. The 20 structures ...詳細: The refinement protocol consisted of slow cooling from 1000 K to 100 K spanning 45 ps. Final structures were energy-minimized using 250 steps of Powell energy minimization. The 20 structures with lowest energy were subjected to further analysis and spin-labeled Cys residues associated with PRE restraints were changed back to their wild type residues. Powell energy minimization was then performed.
NMR constraints
NOE constraints total: 737 / NOE intraresidue total count: 126 / NOE long range total count: 464 / NOE medium range total count: 50 / NOE sequential total count: 97 / Hydrogen bond constraints total count: 72 / Protein chi angle constraints total count: 0 / Protein other angle constraints total count: 0 / Protein phi angle constraints total count: 113 / Protein psi angle constraints total count: 112
代表構造
選択基準: closest to the average
NMRアンサンブル
Average torsion angle constraint violation: 0.2 ° コンフォーマー選択の基準: structures with the lowest energy 計算したコンフォーマーの数: 500 / 登録したコンフォーマーの数: 10 / Maximum lower distance constraint violation: 0.137 Å / Maximum torsion angle constraint violation: 4.569 ° / Maximum upper distance constraint violation: 0.129 Å / Torsion angle constraint violation method: XPLOR
NMR ensemble rms
Distance rms dev: 0.008 Å / Distance rms dev error: 0.0019 Å