2MV6

Solution structure of the transmembrane domain and the juxta-membrane domain of the Erythropoietin Receptor in micelles

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
2	2D 1H-15N HSQC	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
3	3D HNCACB	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
4	3D CBCA(CO)NH	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
5	3D HNCA	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
6	3D HBHA(CO)NH	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
7	3D 1H-15N NOESY	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
8	2D 1H-15N HSQC	20 mM sodium phosphate, 10 v/v D2O, 250 mM DPC, 0.5 mM [U-99% 15N] protein	90% H2O/10% D2O	20	6.5	ambient	313
9	3D HNCO	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313
10	3D HCACO	0.5-1 mM [U-100% 13C; U-100% 15N] protein, 10 % D2O, 20 mM sodium phosphate, 200-400 mM DPC, 1 mM DTT	90% H2O/10% D2O	20	6.5	ambient	313

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	700
2	Bruker	AVANCE	600

NMR Refinement
Method	Details	Software
simulated annealing	simulated annealing was started with 3500 and cool down to 100 K with 15,000 steps. Structure was energy minimized with powell energy minimization.	CYANA

NMR Ensemble Information
Conformer Selection Criteria	structures with the lowest energy
Conformers Calculated Total Number	50
Conformers Submitted Total Number	20
Representative Model	1 (lowest energy)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	structure solution	CYANA		Guntert, Mumenthaler and Wuthrich
2	structure solution	X-PLOR NIH		Schwieters, Kuszewski, Tjandra and Clore
3	processing	NMRPipe		Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
4	peak picking	NMRView		Johnson, One Moon Scientific
5	collection	TopSpin		Bruker Biospin
6	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.