Text: MONOMER BY GEL FILTRATION CHROMATOGRAPHY/LIGHT SCATTERING AND BY NMR.T1/T2(CPMG) (MS/MS) = 921/57 AT 800MHz, TAUC = 9.3(NS). CONSISTENT WITH MOLECULAR WEIGHT.
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試料調製
詳細
Solution-ID
内容
溶媒系
1
0.8 mM [U-100% 13C; U-100% 15N; U-100% 2H] SR10, 10 mM DTT, 0.02 % sodium azide, 5 mM calcium chloride, 100 mM sodium chloride, 1 x protease inhibitor, 20 mM MES, 50 uM DSS, 90% H2O/10% D2O
90% H2O/10% D2O
2
1.2 mM [U-5% 13C; U-100% 15N] SR10, 10 mM DTT, 0.02 % sodium azide, 5 mM calcium chloride, 100 mM sodium chloride, 1 x protease inhibitor, 20 mM MES, 50 uM DSS, 90% H2O/10% D2O
90% H2O/10% D2O
3
1.2 mM [U-5% 13C; U-100% 15N] SR10, 10 mM DTT, 0.02 % sodium azide, 5 mM calcium chloride, 200 mM sodium chloride, 1 x protease inhibitor, 20 mM MES, 50 uM DSS, 90% H2O/10% D2O
90% H2O/10% D2O
試料
濃度 (mg/ml)
構成要素
Isotopic labeling
Solution-ID
0.8mM
SR10-1
[U-100% 13C; U-100% 15N; U-100% 2H]
1
10mM
DTT-2
1
0.02 %
sodium azide-3
1
5mM
calcium chloride-4
1
100mM
sodium chloride-5
1
1 %
protease inhibitor-6
1
20mM
MES-7
1
50uM
DSS-8
1
1.2mM
SR10-9
[U-5% 13C; U-100% 15N]
2
10mM
DTT-10
2
0.02 %
sodium azide-11
2
5mM
calcium chloride-12
2
100mM
sodium chloride-13
2
1 %
protease inhibitor-14
2
20mM
MES-15
2
50uM
DSS-16
2
1.2mM
SR10-17
[U-5% 13C; U-100% 15N]
3
10mM
DTT-18
3
0.02 %
sodium azide-19
3
5mM
calcium chloride-20
3
200mM
sodium chloride-21
3
1 %
protease inhibitor-22
3
20mM
MES-23
3
50uM
DSS-24
3
試料状態
pH: 6.5 / 圧: ambient / 温度: 298 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
800
1
Varian INOVA
Varian
INOVA
600
2
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解析
NMR software
名称
バージョン
開発者
分類
CNS
Brunger, Adams, Clore, Gros, NilgesandRead
refinemen,structuresolution,geometryoptimization
CYANA
3
Guntert, MumenthalerandWuthrich
構造決定
CYANA
3
Guntert, MumenthalerandWuthrich
geometryoptimization
NMRPipe
Delaglio, Grzesiek, Vuister, Zhu, PfeiferandBax
解析
TopSpin
BrukerBiospin
collection
Sparky
Goddard
データ解析
Sparky
Goddard
peakpicking
TALOS+
Shen, Cornilescu, DelaglioandBax
geometryoptimization
PALES
PALES (Zweckstetter, Bax)
geometryoptimization
CYANA
3
Guntert, MumenthalerandWuthrich
精密化
精密化
手法: molecular dynamics / ソフトェア番号: 1 詳細: STRUCTURE DETERMINED BY SPARSE CONSTRAINTS FROM UNIFORMLY DEUTERATED, METHYL BACK-PROTONATED(ILE,VAL,LEU). NOESY ASSIGNMENTS BY CYANA-3.0 WITH RDC. 20 OF 100 STRUCTURES LOWEST TARGET FUNCTION ...詳細: STRUCTURE DETERMINED BY SPARSE CONSTRAINTS FROM UNIFORMLY DEUTERATED, METHYL BACK-PROTONATED(ILE,VAL,LEU). NOESY ASSIGNMENTS BY CYANA-3.0 WITH RDC. 20 OF 100 STRUCTURES LOWEST TARGET FUNCTION SELECTED WITH CYANA-3.0. SELECTED MODELS ARE FURTHER REFINED USING CNS IN EXPLICIT WATER SHELL AND RDC SAMPLE ALIGNED IN PHAGE (NILGES PROTOCOL WITH PARAM19). ASSIGNMENT STATS (ALL RESIDUES INCLUDED): BACKBONE 72.96%, SIDECHAIN 21.00%, AROMATIC (SC) 00.00%, STEREOSPECIFIC VL METHYL ASSIGNMENT 100%, UNAMBIGUOUS SIDECHAIN NH2 57.14%. STRUCTURE BASED ON 603 NOE, 235 DIHE, 85 RDC. MAX NOE VIOLATION: 1.85 A; MAX DIHE VIOLATION: 20.7 DEG. 50 TOTAL CLOSE CONTACTS PER 20 MODELS. STRUCTURE QUALITY FACTOR (PSVS 1.3): ORDERED RESIDUES RANGES: 4-27, 31-33, 37-59, 64-80, 84-95, 98-133, 134-141 FOR [S(PHI)+S(PSI)] > 1.8. SECONDARY STRUCTURE - ALPHA HELICES: 5-22, 126-132, 134-141; BETA STRANDS: 49-53, 40-44, 121-125, 66-67, 113-116, 100-103, 86-90, 77-81 RMSD(ANG): BACKBONE 2.1, ALL -0.45/-1.46 (RAW/Z), PROCHECK (ALL): -0.38/-2.25 (RAW/Z), MOLPROBITY CLASH: 18.61/-1.67 (RAW/Z). RDC STATISTICS FROM CYANA-3.0. DA = -5.404 HZ, RHOM = 0.48; CORR. COEFF: 0.919 +/- 0.012, Q-FACTOR: 25.162 +/- 1.869%. AFTER CNS WATER REFINEMENT WITH RDC PALES COMPUTED CORR. COEFF: 0.976 AND Q-FACTOR: 14.0%.
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