1DC8

STRUCTURE OF A TRANSIENTLY PHOSPHORYLATED "SWITCH" IN BACTERIAL SIGNAL TRANSDUCTION

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	3D_15N-SEPARATED_NOESY	0.3 MM NTRC(1-124) U-15N; 200 MM SODIUM PHOSPHATE, PH 6.75; 50 MM MAGNESIUM CHLORIDE; 200 MM CARBAMOYLPHOSPHATE		250-500 mM	6.75	1 atm	298
2	3D_13C-SEPARATED_NOESY	0.3 MM NTRC(1-124) U-15N,13C; 200 MM SODIUM PHOSPHATE, PH 6.75; 50 MM MAGNESIUM CHLORIDE; 200 MM CARBAMOYLPHOSPHATE		250-500 mM	6.75	1 atm	298
3	2D NOESY	0.3 MM NTRC(1-124) U-15N; 200 MM SODIUM PHOSPHATE, PH 6.75; 50 MM MAGNESIUM CHLORIDE; 200 MM CARBAMOYLPHOSPHATE		250-500 mM	6.75	1 atm	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	DMX	600
2	Bruker	DMX	750

NMR Refinement
Method	Details	Software
torsion angle dynamics	STRUCTURE BASED ON A TOTAL OF 1301 UNIQUE DISTANCE CONSTRAINTS (OBTAINED FROM 3095 NOE CROSSPEAKS), INCLUDING 374 INTRARESIDUE, 405 SHORT-RANGE, 228 MEDIUM RANGE AND 294 LONG-RANGE CONSTRAINTS. DYANA 1.5 ANNEAL COMMAND (10000 STEPS) USED TO GENERATE 40 CONFORMERS. 20 LOWEST TARGET FUNCTION STRUCTURES ANALYZED. CONFORMER 4 CHOSEN FOR DEPOSITION AS CLOSEST TO MEAN COORDINATES OF THE ENSEMBLE.	DYANA

NMR Ensemble Information
Conformer Selection Criteria
Conformers Calculated Total Number
Conformers Submitted Total Number	1
Representative Model	(closest to the average)

Additional NMR Experimental Information
Details	PHOSPHORYLATION (>95%) COULD BE MAINTAINED FOR 36 HR. TO OBTAIN SUFFICIENT S/N IN 3D NOESY EXPERIMENTS, MULTIPLE (TYPICALLY 4) COMPLETE 3D DATASETS WERE ACQUIRED ON FRESHLY PREPARED SAMPLES AND THEN COADDED TO YIELD HIGH-QUALITY DATASETS FOR STRUCTURE DETERMINATION.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	structure solution	DYANA	1.5	PETER GUENTERT
2	processing	Felix	95.0	MSI
3	collection	XwinNMR	1.5	BRUKER
4	data analysis	DYANA	1.5	PETER GUENTERT
5	data analysis	XEASY	1.3.13	MUMENTHALER
6	refinement	DYANA	1.5	PETER GUENTERT

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.