2EU0

The NMR ensemble structure of the Itk SH2 domain bound to a phosphopeptide

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D NOESY	1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
2	3D_15N-separated_NOESY	U-15N labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
3	HNHA	U-15N labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
4	3D_13C-separated_NOESY	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
5	3D_13C_separated_aromatic_NOESY	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
6	HNCACB	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
7	CBCA(CO)NH	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
8	HCCH_TOCSY	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
9	CCONH	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
10	13C edited_13C/15_N filtered NOESY	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
11	15N_edited_13C/15N_filtered NOESY	U-15N, 13-C labeled, 1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90% H2O/10% D2O	125mM	7.4	ambient	298
12	2D TOCSY	5mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	90%H20, 10%D2O	125mM	7.4	ambient	298
13	D2O exchange experiment	1mM Itk SH2 domain, 20mM phosphopeptide, 50mM KH2PO4, 75mM NaCl, 2mM DTT, 0.02% NaN3, pH 7.4	100% D2O	125mM	7.4	ambient	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	DRX	500

NMR Refinement
Method	Details	Software
Distance geometry simulated annealing was used for refinement	2486 Intramolecular NOE restraints, 31 intermolecular NOE restraints, 63 dihedral angles calculated using HNHA experiment and TALOS and 27 hydrogen bonds (D20 exchange based restraints)	XwinNMR

NMR Ensemble Information
Conformer Selection Criteria	structures with the least restraint violations
Conformers Calculated Total Number	200
Conformers Submitted Total Number	20
Representative Model	1 (lowest energy)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	collection	XwinNMR
2	processing	NMRPipe		Delaglio et al
3	data analysis	NMRView	4.1.3	Johnson and Blevins
4	refinement	CNS	1.0	Brunger et al

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.