2L02

Solution NMR Structure of protein BT2368 from Bacteroides thetaiotaomicron, Northeast Structural Genomics Consortium Target BtR375

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
2	2D 1H-13C CT-HSQC aliphatic	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
3	3D HNCO	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
4	3D CBCA(CO)NH	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
5	3D HNCACB	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
6	2D 1H-13C CT-HSQC aromatic	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
7	3D simutaneous 13C-aromatic,13C-aliphatic,15N edited 1H-1H NOESY	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
8	3D HN(CA)CO	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
9	3D HBHA(CO)NH	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
10	3D HCCH-COSY aliphatic	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
11	3D HCCH-COSY aromatic	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
12	3D HCCH-TOCSY aliphatic	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
13	2D 1H-13C CT-HSQC methyl	1.1 mM [U-5% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
14	2D J-modulation 1H-15N HSQC	1.1 mM [U-5% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298
15	2D J-modulation 1H-15N HSQC	0.9 mM [U-5% 13C; U-100% 15N] BtR375, 18 mM ammonium acetate, 91 mM sodium chloride, 4.6 mM calcium chloride, 0.018 % sodium azide, 4 % C12E5 polyethylene glycol, 4 % hexanol	80% H2O/20% D2O		6.5	ambient	298
16	2D J-modulation 1H-15N HSQC	1.2 mM [U-5% 13C; U-100% 15N] BtR375, 18 mM ammonium acetate, 91 mM sodium chloride, 4.6 mM calcium chloride, 0.018 % sodium azide	80% H2O/20% D2O		6.5	ambient	298
17	2D 1H-15N LR-HSQC for Histidine	1.1 mM [U-100% 13C; U-100% 15N] BtR375, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 0.02 % sodium azide	90% H2O/10% D2O		6.5	ambient	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	600
2	Varian	INOVA	600
3	Varian	INOVA	750
4	Varian	INOVA	600

NMR Refinement
Method	Details	Software
simulated annealing	Structure determination was performed iteratively with CYANA 3.0 using NOE-based constraints, PHI and PSI dihedral angle constraints from TALOS+, and backbone H-N residual dipolar couplings from two alignment media. 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 and upper limit constraints relaxed by 5%	CNS

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

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinement	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
2	structure solution	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
3	geometry optimization	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
4	refinement	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
5	geometry optimization	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
6	structure solution	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
7	data analysis	AutoStructure	2.2.1	Huang, Tejero, Powers and Montelione
8	data analysis	AutoAssign	2.3.0	Zimmerman, Moseley, Kulikowski and Montelione
9	chemical shift assignment	AutoAssign	2.3.0	Zimmerman, Moseley, Kulikowski and Montelione
10	processing	NMRPipe		Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
11	data analysis	XEASY	1.3.13	Bartels et al.
12	collection	TopSpin	2.1	Bruker Biospin
13	processing	TopSpin	2.1	Bruker Biospin
14	collection	VnmrJ	2.1B	Varian
15	geometry optimization	TALOS+	1.2009.0721.18	Shen, Cornilescu, Delaglio and Bax
16	chemical shift assignment	CARA	1.8.4	Keller and Wuthrich
17	data analysis	CARA	1.8.4	Keller and Wuthrich
18	peak picking	CARA	1.8.4	Keller and Wuthrich
19	processing	PROSA	6.4	Guntert

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), 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.

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2L02

Solution NMR Structure of protein BT2368 from Bacteroides thetaiotaomicron, Northeast Structural Genomics Consortium Target BtR375