2Z33

Solution structure of the DNA complex of PhoB DNA-binding/transactivation Domain

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	3D SEQUENTIAL ASSIGNMENT PROTOCOL	1-2mM PhoB-DNA U-15N, 13C; 10mM potassium phosphate buffer; 90% H2O, 10% D2O	90% H2O/10% D2O		6.8	AMBIENT	310
2	3D HNHA	1-2mM PhoB-DNA U-15N, 13C; 10mM potassium phosphate buffer; 90% H2O, 10% D2O	90% H2O/10% D2O		6.8	AMBIENT	310
3	4D_13C-SEPARATED_NOESY	1-2mM PhoB-DNA U-15N, 13C; 10mM potassium phosphate buffer; 90% H2O, 10% D2O	90% H2O/10% D2O		6.8	AMBIENT	310
4	4D_13C/15N-SEPARATED_NOESY	1-2mM PhoB-DNA U-15N, 13C; 10mM potassium phosphate buffer; 90% H2O, 10% D2O	90% H2O/10% D2O		6.8	AMBIENT	310
5	3D_15N-SEPARATED_NOESY	1-2mM PhoB-DNA U-15N, 13C; 10mM potassium phosphate buffer; 90% H2O, 10% D2O	90% H2O/10% D2O		6.8	AMBIENT	310
6	3D_13C_SEPARATED_NOESY, 2D NOESY	1-2mM PhoB-DNA U-15N, 13C; 10mM potassium phosphate buffer; 90% H2O, 10% D2O	90% H2O/10% D2O		6.8	AMBIENT	310
7	2D TOCSY AND 2D COSY	1-2mM PhoB-DNA U-15N, 13C; 10mM potassium phosphate buffer; 90% H2O, 10% D2O	90% H2O/10% D2O		6.8	AMBIENT	310

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	DRX	600
2	Bruker	DRX	500

NMR Refinement
Method	Details	Software
simulated annealing molecluar dynamics	THE STRUCTURES ARE BASED ON A TOTAL OF 2508 RESTRAINTS, 2263 ARE NOE-DERIVED DISTANCE RESTRAINTS 229 ARE DIHEDRAL ANGLE RESTRAINTS AND 16 ARE RESTRAINTS OF DNA BASE PLANALITY RESTRAINTS. STRUCTURE CALCULATIONS WERE PERFORMED WITH CNS BY FOLLOWIN PROCEDURE. FIRST, ONLY THE STRUCTURES OF PROTEIN WERE CALCULATED USING THE HYBRID DISTANCE GEOMETRY-SIMULATED ANNEAING PROTOCOL WITH THE INTRA-PROTEIN RESTRAINTS, 10 OUT OF 100 CALCULATED STRUCTURES WERE SELECTED BY HAVING NO DISTANCE VIOLATION GREATER THAN 0.2 ANGSTROMS AND NO DIHEDRAL ANGLE VIOLATION GREATER THAN 2 DEGREES and A LOW TARGET ENERGY. SECOND, THE PROTEIN AND AN IDEALIZED B-FORM OF 16-MER DUPLEX DNA WERE DOCKED BY A SIMULATED ANNEALING PROTOCOL, FOR WHICH THE INTRA-DNA, INTER-MOLECULAR AND INTRA-PROTEIN RESTRAINTS WERE EMPLOYED. AS AN INITIAL STRUCTURE, EACH OF THE 10 PROTEIN STRUCTURES WAS PLACED IN 15 DIFFERENT POSITIONS 50 ANGSTROMS AWAY FROM THE DNA. NEXT, EACH OF THE 150 DOCKED COMPLEX STRUCTURES WAS FURTHER REFINED BY A SIMULATED ANNEALING PROTOCOL. FINALLY, 20 STRUCTURES WERE SELECTED BY HAVING NO DISTANCE VIOLATION GREATER THAN 0.2 ANGSTROME AND NO TORSION ANGLE VIOLATION THAN 2 DEGREES AND A LOW TARGET ENERGY. ADDITIONALLY, 10 CNS-SELECTED STRUCTURES WERE REFINED IN REALISTIC SOLUTION SISTEM BY USING MOLECULAR DYNAMICS SIMULATION PROGRAM, MARBLE WITH RESTRAINT FUNCTIONS FOR NOE DISTANCE DIHEDRAL ANGLE AND PLANARITY OF DNA BASE PAIR USED IN CNS.	NMRPipe

NMR Ensemble Information
Conformer Selection Criteria	structures with the least restraint violations, structures with the lowest energy
Conformers Calculated Total Number	100
Conformers Submitted Total Number	10
Representative Model	1 (closest to the average)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	data analysis	NMRPipe
2	data analysis	PIPP
3	refinement	CNS	1.0	A.BRUNGER et al.
4	refinement	MARBLE	0.3.46	M.Ikeguchi

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.