Experiment: 2F33

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	4D_13C/15N-separated_NOESY	1mM calbindin D28k; U-15N,13C; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2, 90% H2O, 10% D2O	90% H2O/10% D2O	6 mM CaCl2	6.2	ambient	323
2	4D_13C/13C-separated_NOESY	1mM calbindin D28k; U-15N,13C; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2	100% D2O	6 mM CaCl2	6.2	ambient	323
3	3D_CT_13C_methyl-separated_NOESY	1mM calbindin D28k; U-2H,15N,13C; Ile (d1 only), Leu, Val methyl protonated; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2, 90% H2O, 10% D2O	90% H2O/10% D2O	6 mM CaCl2	7.0	ambient	298
4	(4,2)D_CT_13C/13C_methyl-separated_NOESY	1mM calbindin D28k; U-2H,15N,13C; Ile (d1 only), Leu, Val methyl protonated; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2, 90% H2O, 10% D2O	90% H2O/10% D2O	6 mM CaCl2	6.2	ambient	323
5	4D_13C/15N-separated_NOESY	1mM calbindin D28k; U-2H,15N,13C; Ile (d1 only), Leu, Val methyl protonated; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2, 90% H2O, 10% D2O	90% H2O/10% D2O	6 mM CaCl2	7.0	ambient	298
6	(4,2)D_13C/15N-separated_NOESY	1mM calbindin D28k; U-2H,15N,13C; Ile (d1 only), Leu, Val methyl protonated; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2, 90% H2O, 10% D2O	90% H2O/10% D2O	6 mM CaCl2	6.2	ambient	323
7	4D_15N/15N-separated_NOESY	1mM calbindin D28k; U-2H,15N; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2, 90% H2O, 10% D2O	90% H2O/10% D2O	6 mM CaCl2	7.0	ambient	298
8	(4,2)D_13C_ali/13C_aro-separated_NOESY	1mM calbindin D28k; U-15N,13C; 10 mM Tris, 1 mM DTT, 0.02% NaN3, 6 mM CaCl2, 90% H2O, 10% D2O	90% H2O/10% D2O	6 mM CaCl2	6.2	ambient	323

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

NMR Refinement
Method	Details	Software
torsion angle dynamics molecular dynamics simulated annealing	the structures are based on 6882 NOE distance constraints, 432 dihedral angle restraints, 36 hydrogen bond restraints and 304 residual dipolar coupling restraints	CNS

NMR Ensemble Information
Conformer Selection Criteria	structures with the least restraint violations,structures with the lowest energy
Conformers Calculated Total Number	900
Conformers Submitted Total Number	10
Representative Model	1 (fewest violations,lowest energy)

Additional NMR Experimental Information
Details	This structure was determined using a combination of 3D, 4D and (4,2)D projection reconstruction techniques on perdeuterated and fully protonated samples.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinement	CNS	1.0	Brunger, A.T., Adams, P.D., Clore, G.M., Delano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T., Warren, G.L.
2	processing	NMRPipe	2.3	Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., Bax, A.
3	data analysis	NMRView	5.0	Johnson, B.A., Blevins, R.A.

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.