Experiment: 2K27

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
2	2D 1H-13C HSQC CT	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
3	3D CBCA(CO)NH	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
4	3D C(CO)NH	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
5	3D HNCO	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
6	3D HNCA	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
7	3D HNCACB	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
8	3D HN(CO)CA	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
9	3D HCCH-TOCSY	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
10	3D 1H-15N NOESY	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
11	3D 1H-13C NOESY	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
12	3D CBCANH	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00
13	3D HN(COCA)CB	0.9 mM [U-100% 13C; U-100% 15N] Pax8 Paired Box Domain, 40 uM DSS, 50 mM sodium phosphate, 0.1 % sodium azide, 15 mM [U-100% 2H] DTT	90% H2O/10% D2O	50	6.2	ambient	298.00

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	500
2	Bruker	Avance 2	750

NMR Refinement
Method	Details	Software
molecular mechanics	Minimization was performed by means of Discover, with 200 iterations using the steep descent and 800 iterations using the conjugate gradients algorithms. c-terminal superposition is performed between residues 87-135 and the n-terminal superposition can be performed between 29-62.	CYANA

NMR Ensemble Information
Conformer Selection Criteria	structures with the lowest energy
Conformers Calculated Total Number	300
Conformers Submitted Total Number	20
Representative Model	1 (lowest energy)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	structure solution	CYANA	2.1	Guntert, Mumenthaler and Wuthrich
2	data analysis	TALOS	(NMR-Pipe 3.5)	Cornilescu, Delaglio and Bax
3	validation	ProcheckNMR	3.4	Laskowski and MacArthur
4	collection	TopSpin	1.3	Bruker Biospin
5	processing	TopSpin	1.3	Bruker Biospin
6	chemical shift assignment	CARA	1.8.4	Rochus Keller
7	data analysis	CARA	1.8.4	Rochus Keller
8	peak picking	CARA	1.8.4	Rochus Keller
9	refinement	Discover	2.98	Accelrys Software Inc.
10	validation	AQUA	3.2	Rullmann, Doreleijers and Kaptein
11	validation	WHAT IF		Vriend

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.