手法: 溶液NMR 詳細: NMR solution structure of the complex between a MAP1LC3 protein, GABARAPL-1, and the LIR motif of NBR1
NMR実験
Conditions-ID
Experiment-ID
Solution-ID
タイプ
1
1
1
2D 1H-15N HSQC
1
2
3
2D 1H-15N HSQC
1
3
2
2D 1H-13C HSQC
1
4
4
2D 1H-13C HSQC aliphatic
1
5
4
2D 1H-13C HSQC aromatic
1
6
4
2D 1H-13C HSQC
1
7
2
3D HNCO
1
8
2
3DHN(CA)CO
1
9
2
3D HNCA
1
10
2
3D HN(CA)CB
1
11
2
3D 1H-15N NOESY
1
12
2
3D 1H-13C NOESY
1
13
2
TROSY-H(CCCO)NH-TOCSY
1
14
2
(H)CC(CO)NH-TOCSY
1
15
4
3D HNCA
1
16
4
3D HN(CA)CB
1
17
4
3D 1H-15N NOESY
1
18
4
3D 1H-13C NOESY
1
19
4
3D (H)CCH-TOCSY
1
20
4
TROSY-H(CCCO)NH-TOCSY
1
21
4
(H)CC(CO)NH-TOCSY
-
試料調製
詳細
Solution-ID
内容
溶媒系
1
0.6 mM [U-98% 15N] GABARAPL-1, 0.9 mM NBR1-LIR, 50 mM sodium phosphate, 100 mM sodium chloride, 4.6 mM sodium azide, 1 mM protease inhibitor cocktail, 0.3 mM DSS, 95% H2O/5% D2O
95% H2O/5% D2O
2
0.6 mM [U-98% 13C; U-98% 15N] GABARAPL-1, 0.9 mM NBR1-LIR, 50 mM sodium phosphate, 100 mM sodium chloride, 4.6 mM sodium azide, 1 mM protease inhibitor cocktail, 0.3 mM DSS, 95% H2O/5% D2O
95% H2O/5% D2O
3
0.9 mM GABARAPL-1, 0.6 mM [U-98% 15N] NBR1-LIR, 50 mM sodium phosphate, 100 mM sodium chloride, 4.6 mM sodium azide, 1 mM protease inhibitor cocktail, 0.3 mM DSS, 95% H2O/5% D2O
95% H2O/5% D2O
4
0.9 mM GABARAPL-1, 0.6 mM [U-98% 13C; U-98% 15N] NBR1-LIR, 50 mM sodium phosphate, 100 mM sodium chloride, 4.6 mM sodium azide, 1 mM protease inhibitor cocktail, 0.3 mM DSS, 95% H2O/5% D2O
95% H2O/5% D2O
試料
濃度 (mg/ml)
構成要素
Isotopic labeling
Solution-ID
0.6mM
GABARAPL-1-1
[U-98% 15N]
1
0.9mM
NBR1-LIR-2
1
50mM
sodium phosphate-3
1
100mM
sodium chloride-4
1
4.6mM
sodium azide-5
1
1mM
protease inhibitor cocktail-6
1
0.3mM
DSS-7
1
0.6mM
GABARAPL-1-8
[U-98% 13C; U-98% 15N]
2
0.9mM
NBR1-LIR-9
2
50mM
sodium phosphate-10
2
100mM
sodium chloride-11
2
4.6mM
sodium azide-12
2
1mM
protease inhibitor cocktail-13
2
0.3mM
DSS-14
2
0.9mM
GABARAPL-1-15
3
0.6mM
NBR1-LIR-16
[U-98% 15N]
3
50mM
sodium phosphate-17
3
100mM
sodium chloride-18
3
4.6mM
sodium azide-19
3
1mM
protease inhibitor cocktail-20
3
0.3mM
DSS-21
3
0.9mM
GABARAPL-1-22
4
0.6mM
NBR1-LIR-23
[U-98% 13C; U-98% 15N]
4
50mM
sodium phosphate-24
4
100mM
sodium chloride-25
4
4.6mM
sodium azide-26
4
1mM
protease inhibitor cocktail-27
4
0.3mM
DSS-28
4
試料状態
イオン強度: 0.1 / pH: 7 / 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
Bruker Avance
Bruker
AVANCE
900
1
Bruker Avance
Bruker
AVANCE
800
2
Bruker Avance
Bruker
AVANCE
700
3
Bruker Avance
Bruker
AVANCE
600
4
-
解析
NMR software
名称
開発者
分類
TopSpin
BrukerBiospin
collection
TopSpin
BrukerBiospin
解析
Sparky
Goddard
chemicalshiftassignment
Sparky
Goddard
データ解析
Sparky
Goddard
peakpicking
TALOS
Cornilescu, DelaglioandBax
構造決定
CSI
Wishart, D. S. & Sykes, B. D.
構造決定
CYANA
Guntert, MumenthalerandWuthrich
構造決定
CYANA
Guntert, MumenthalerandWuthrich
精密化
OPAL
Luginbuhl, Guntert, BilleterandWuthrich
精密化
精密化
手法: energy minimization / ソフトェア番号: 1
NMR constraints
NOE constraints total: 1448 / NOE intraresidue total count: 207 / NOE long range total count: 510 / NOE medium range total count: 245 / NOE sequential total count: 436 / Hydrogen bond constraints total count: 84
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: 20 / Maximum upper distance constraint violation: 0.09 Å
NMR ensemble rms
Distance rms dev: 0.0081 Å / Distance rms dev error: 0.0004 Å