Experiment: 2RP3

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	1.5mM [U-100% 15N] CVNmutDB, 1.5mM [U-100% 13C; U-100% 15N] CVNmutDB	90% H2O/10% D2O		6.0	atm	303
2	3D CBCA(CO)NH	1.5mM [U-100% 15N] CVNmutDB, 1.5mM [U-100% 13C; U-100% 15N] CVNmutDB	90% H2O/10% D2O		6.0	atm	303
3	3D HNCACB	1.5mM [U-100% 15N] CVNmutDB, 1.5mM [U-100% 13C; U-100% 15N] CVNmutDB	90% H2O/10% D2O		6.0	atm	303
4	3D HCCH-TOCSY	1.5mM [U-100% 15N] CVNmutDB, 1.5mM [U-100% 13C; U-100% 15N] CVNmutDB	90% H2O/10% D2O		6.0	atm	303
5	3D 15N-NOESY HSQC	1.5mM [U-100% 15N] CVNmutDB, 1.5mM [U-100% 13C; U-100% 15N] CVNmutDB	90% H2O/10% D2O		6.0	atm	303
6	13C-NOESY	1.5mM [U-100% 15N] CVNmutDB, 1.5mM [U-100% 13C; U-100% 15N] CVNmutDB	90% H2O/10% D2O		6.0	atm	303

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

NMR Refinement
Method	Details	Software
simulated annealing	BACKBONE NH DIPOLAR COUPLINGS, RMS=0.64HZ	CYANA

NMR Ensemble Information
Conformer Selection Criteria	structures with the lowest energy
Conformers Calculated Total Number	50
Conformers Submitted Total Number	20
Representative Model	1 (lowest energy)

Additional NMR Experimental Information
Details	THE INITIAL STRUCTURES WERE OBTAINED USING CYANA AUTOMATIC CALCULATION, BASED ON CHEMICAL SHIFT LISTS FROM SEQUENCE-SPECIFIC RESONANCE ASSIGNMENT AND NOES FROM 15N AND 13C-EDITED 3D-NOESY SPECTRA. THROUGHOUT ALL CALCULATIONS, 126 BACKBONE TORSION ANGLE CONSTRAINTS DERIVED FROM TALOS, WERE EMPLOYED. CNS WAS USED FOR FURTHER REFINEMENT, USING THE DISTANCE AND DIHEDRAL ANGLE CONSTRAINTS OBTAINED FROM THE FINAL CYCLE OF THE CYANA CALCULATION, AND SEVERAL ADDITIONAL NOE CONSTRAINTS FROM MANUAL CHECKING OF THE 3D NOESY DATA. IN TOTAL, 2076 EXPERIMENTAL NOE-RESTRAINTS (~20 PER RESIDUE) WERE EMPLOYED. FROM NOE-DERIVED ENSEMBLE OF 50 STRUCTURES THE 20 LOWEST ENERGY STRUCTURES WERE FURTHER REFINED AGAINST 15N-1H RESIDUAL DIPOLAR COUPLINGS (62) WITH THE PROGRAM DYNAMO INCLUDED IN THE NMRPIPE PACKAGE.

Additional NMR Experimental Information

Details

THE INITIAL STRUCTURES WERE OBTAINED USING CYANA AUTOMATIC CALCULATION, BASED ON CHEMICAL SHIFT LISTS FROM SEQUENCE-SPECIFIC RESONANCE ASSIGNMENT AND NOES FROM 15N AND 13C-EDITED 3D-NOESY SPECTRA. THROUGHOUT ALL CALCULATIONS, 126 BACKBONE TORSION ANGLE CONSTRAINTS DERIVED FROM TALOS, WERE EMPLOYED. CNS WAS USED FOR FURTHER REFINEMENT, USING THE DISTANCE AND DIHEDRAL ANGLE CONSTRAINTS OBTAINED FROM THE FINAL CYCLE OF THE CYANA CALCULATION, AND SEVERAL ADDITIONAL NOE CONSTRAINTS FROM MANUAL CHECKING OF THE 3D NOESY DATA. IN TOTAL, 2076 EXPERIMENTAL NOE-RESTRAINTS (~20 PER RESIDUE) WERE EMPLOYED. FROM NOE-DERIVED ENSEMBLE OF 50 STRUCTURES THE 20 LOWEST ENERGY STRUCTURES WERE FURTHER REFINED AGAINST 15N-1H RESIDUAL DIPOLAR COUPLINGS (62) WITH THE PROGRAM DYNAMO INCLUDED IN THE NMRPIPE PACKAGE.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinement	CYANA		HERRMANN, GUNTERT, WUTHRICH
2	refinement	CNS		BRUNGER, ADAMS, CLORE, DELANO, GROSSE-KUNSTLEVE, JIANG, KUSZEWSKI, NILGES, PANNU, READ, RICE, SIMONSON, WARREN
3	structure solution	TopSpin		Bruker Biospin
4	structure solution	NMRPipe		Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
5	structure solution	NMRView		Johnson, One Moon Scientific
6	structure solution	CYANA		Guntert, Mumenthaler and Wuthrich
7	structure solution	TALOS		Cornilescu, Delaglio and Bax
8	structure solution	DYNAMO(NMRpipe)		Cornilescu, Delaglio and Bax

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.

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2RP3

Solution Structure of Cyanovirin-N Domain B Mutant