Experiment: 2M4M

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	15N HSQC	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298
2	15N NOESY-HSQC	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298
3	13C HSQC	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA	100% D2O	110	4.5	1 atm	298
4	aromatic 13C HSQC	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA	100% D2O	110	4.5	1 atm	298
5	13C NOESY-HSQC	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA	100% D2O	110	4.5	1 atm	298
6	13C aromatic NOESY-HSQC	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA	100% D2O	110	4.5	1 atm	298
7	HNCO	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298
8	HNCACO	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298
9	HNCA	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298
10	HNCOCA	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298
11	HNCACB	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298
12	CBCACONH	1.0 mM [U-100% 13C; U-100% 15N] RRM domain of the hypothetical protein, 10 mM sodium acetate, 100 mM sodium chloride, 0.5 mM DTT, 0.5 mM EDTA, 90% H2O, 10% D2O	90% H2O/10% D2O	110	4.5	1 atm	298

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

NMR Refinement
Method	Details	Software
simulating annealing	Refinement in a box of water	CNS

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

Additional NMR Experimental Information
Details	All 3D experiments were acquired with 30% non-uniform sampling using the MDDNMR approach

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinement	CNS	1.21	Brunger A. T. et.al.
2	collection	VnmrJ	2.2D	Varian
3	collection	TopSpin	2.1	Bruker Biospin
4	collection	MddNMR	2.2	(MDDNMR) Orekhov, Jaravine, Kazimierczuk
5	processing	MddNMR	2.2	(MDDNMR) Orekhov, Jaravine, Kazimierczuk
6	data analysis	CCPN_Analysis	2.2	CCPN
7	data analysis	ARIA	2.3	Linge, O'Donoghue and Nilges
8	structure solution	X-PLOR NIH	2.32	Schwieters, Kuszewski, Tjandra and Clore
9	refinement	X-PLOR NIH

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.