2KZN

Solution NMR Structure of Peptide methionine sulfoxide reductase msrB from Bacillus subtilis, Northeast Structural Genomics Consortium Target SR10

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	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		6.5	ambient	298
2	2D 1H-13C HSQC	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		6.5	ambient	298
3	3D HNCO	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		6.5	ambient	298
4	3D HNCA	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		6.5	ambient	298
5	3D TRHNCACB 2H dec	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		6.5	ambient	298
6	3D HN(CA)CO	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		6.5	ambient	298
7	2D 1H-15N HSQC - T1	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		6.5	ambient	298
8	2D 1H-15N HSQC - T2	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		6.5	ambient	298
9	3D 1H-15N NOESY	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		6.5	ambient	298
10	3D 1H-13C NOESY	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		6.5	ambient	298
11	3D (N15)HSQC-NOESY-(N15)HSQC	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		6.5	ambient	298
12	3D (C13)HSQC-NOESY-(C13)HSQC	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		6.5	ambient	298
13	3D (N15)HSQC-NOESY-(C13)HSQC	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		6.5	ambient	298
14	3D (C13)HSQC-NOESY-(N15)HSQC	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		6.5	ambient	298
15	2D 1H-13C HSQC	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		6.5	ambient	298
16	2D 1H-15N TROSY	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		6.5	ambient	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	800
2	Varian	INOVA	600

NMR Refinement
Method	Details	Software
molecular dynamics	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%.	CNS

NMR Ensemble Information
Conformer Selection Criteria	target function
Conformers Calculated Total Number	100
Conformers Submitted Total Number	20
Representative Model	1 (lowest energy)

Additional NMR Experimental Information
Details	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.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinemen,structure solution,geometry optimization	CNS		Brunger, Adams, Clore, Gros, Nilges and Read
2	structure solution	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
3	geometry optimization	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
4	processing	NMRPipe		Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
5	collection	TopSpin		Bruker Biospin
6	data analysis	Sparky		Goddard
7	peak picking	Sparky		Goddard
8	geometry optimization	TALOS+		Shen, Cornilescu, Delaglio and Bax
9	geometry optimization	PALES		PALES (Zweckstetter, Bax)
10	refinement	CYANA	3.0	Guntert, Mumenthaler and Wuthrich

RCSB PDB Core Operations are funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.