2LY9

Solution NMR Structure of Homeobox 2 Domain from Human ZHX1 repressor, Northeast Structural Genomics Consortium (NESG) Target HR7907F

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
2	2D 1H-13C HSQC	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
3	3D HNCO	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
4	(4,3)D GFT CABCA(CO)NHN	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
5	(4,3)D GFT HNNCABCA	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
6	2D 1H-13C HSQC	0.3 mM [5% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
7	(4,3)D GFT HCCH-COSY alipahtic	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
8	3D (H)CCH-TOCSY aliphatic	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
9	3D 13C/15N-edited NOESY	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
10	(4,3)D GFT HABCAB(CO)NH	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298
11	3D (H)CCH-COSY aromatic	0.4 mM [U-100% 13C; U-100% 15N] HR7907F, 0.02 % sodium azide, 10 mM DTT, 5 mM calcium chloride, 100 mM sodium chloride, 1 x Proteinase Inhibitors, 20 mM MES, 50 uM DSS	90% H2O/10% D2O		6.5	1 atm	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Varian	INOVA	600
2	Varian	INOVA	750

NMR Refinement
Method	Details	Software
simulated annealing	STRUCTURE DETERMINATION WAS PERFORMED BY RUNNING CYANA AND ASDP IN PARALLEL USING NOE-BASED CONSTRAINTS AND PHI AND PSI DIHEDRAL ANGLE CONSTRAINTS FROM TALOS+. CONSENSUS PEAK ASSIGNMENTS WERE SELECTED AND USED IN ITERATIVE REFINEMENT WITH CYANA. THE 20 CONFORMERS OUT OF 100 WITH THE LOWEST TARGET FUNCTION WERE FURTHER REFINED BY SIMULATED ANNEALING IN EXPLICIT WATER BATH USING THE PROGRAM CNS WITH PARAM19 FORCE FIELD NMR ENSEMBLE INFORMATION	CARA

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

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	peak picking	CARA	1.8.4	Keller and Wuthrich
2	chemical shift assignment	CARA	1.8.4	Keller and Wuthrich
3	refinement	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
4	geometry optimization	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
5	structure solution	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
6	data analysis	AS-DP	1.0	Huang, Tejero, Powers and Montelione
7	refinement	AS-DP	1.0	Huang, Tejero, Powers and Montelione
8	data analysis	AutoAssign	2.3.0	Zimmerman, Moseley, Kulikowski and Montelione
9	chemical shift assignment	AutoAssign	2.3.0	Zimmerman, Moseley, Kulikowski and Montelione
10	data analysis	XEASY		Bartels et al.
11	collection	VnmrJ		Varian
12	geometry optimization	TALOS+		Shen, Cornilescu, Delaglio and Bax
13	structure validation	PSVS		Bhattacharya, Montelione

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.