2LJW

Solution NMR structure of Alr2454 protein from Nostoc sp. strain PCC 7120, Northeast Structural Genomics Consortium Target NsR264

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
2	2D 1H-13C HSQC	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
3	3D 1H-15N NOESY	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
4	3D 1H-13C NOESY aliphatic	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
5	3D 1H-13C NOESY aromatic	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
6	2D 1H-13C HSQC high resolution (L/V methyl stereoassignment)	0.567 mM [U-5% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
7	3D HNCO	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
8	3D HN(CA)CO	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
9	3D HNCA	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
10	3D HN(CO)CA	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
11	3D CBCA(CO)NH	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
12	3D HNCACB	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
13	3D HBHA(CO)NH	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
14	3D HCCH-TOCSY	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
15	3D CCH-TOCSY	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
16	3D HCCH-COSY	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
17	1D 15N T1 and T2	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298
18	2D 1H-15N hetNOE	1.01 mM [U-100% 13C; U-100% 15N] NsR264, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O		4.5	ambient	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	800
2	Bruker	AVANCE	600

NMR Refinement
Method	Details	Software
simulated annealing	THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2478 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 162 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (25.4 CONSTRAINTS PER RESIDUE, 6.8 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 104 BY PSVS 1.4). STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 3.0. THE 20 STRUCTURES OUT OF 100 WITH THE LOWEST TARGET FUNCTION WERE FURTHER REFINED BY RESTRAINED MOLECULAR DYNAMICS/ENERGY MINIMIZATION IN EXPLICIT WATER (CNS) WITH PARAM19.	CNS

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

Additional NMR Experimental Information
Details	THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. SPECTRA FOR BACKBONE AND SIDE CHAIN ASSIGNMENTS WERE OBTAINED ON A 1.7-MM MICROCRYOPROBE AT 600 MHZ. ALL NOESY DATA WERE ACQUIRED AT 800 MHZ USING A 5-MM CRYOPROBE. BACKBONE ASSIGNMENTS WERE MADE USING PINE, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 3.0. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOSplus. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HIS6): BACKBONE, 99.4%, SIDE CHAIN, 98.3%, AROMATICS, 96.6%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 100.0%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 104, PSVS 1.4), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 1-100: (A) RMSD (ORDERED RESIDUES): BB, 0.6, HEAVY ATOM, 0.9. (B) MOLPROBITY RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 96.8%, ADDITIONALLY ALLOWED, 3.1%, DISALLOWED, 0.1%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.15/-0.28, ALL, -0.03/-0.18. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 12.51/-0.62 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1 TO 104): RECALL, 0.976, PRECISION, 0.934, F-MEASURE, 0.955, DP-SCORE, 0.817. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 2. THE FINAL SIX HISTIDINE RESIDUES IN THE C-TERMINAL AFFINITY TAG WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION.

Additional NMR Experimental Information

Details

THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. SPECTRA FOR BACKBONE AND SIDE CHAIN ASSIGNMENTS WERE OBTAINED ON A 1.7-MM MICROCRYOPROBE AT 600 MHZ. ALL NOESY DATA WERE ACQUIRED AT 800 MHZ USING A 5-MM CRYOPROBE. BACKBONE ASSIGNMENTS WERE MADE USING PINE, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 3.0. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOSplus. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HIS6): BACKBONE, 99.4%, SIDE CHAIN, 98.3%, AROMATICS, 96.6%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 100.0%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 104, PSVS 1.4), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 1-100: (A) RMSD (ORDERED RESIDUES): BB, 0.6, HEAVY ATOM, 0.9. (B) MOLPROBITY RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 96.8%, ADDITIONALLY ALLOWED, 3.1%, DISALLOWED, 0.1%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.15/-0.28, ALL, -0.03/-0.18. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 12.51/-0.62 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1 TO 104): RECALL, 0.976, PRECISION, 0.934, F-MEASURE, 0.955, DP-SCORE, 0.817. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 2. THE FINAL SIX HISTIDINE RESIDUES IN THE C-TERMINAL AFFINITY TAG WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinement	CNS	1.3	Brunger, Adams, Clore, Gros, Nilges and Read
2	refinement, structure solution	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
3	rpf analysis	AutoStructure	2.2.1	Huang, Tejero, Powers and Montelione
4	processing	NMRPipe	2.3	Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
5	collection	TopSpin	2.1	Bruker Biospin
6	data analysis	TopSpin	2.1	Bruker Biospin
7	chemical shift assignment	PINE	1.0	Bahrami, Markley, Assadi, and Eghbalnia
8	data analysis	Sparky	3.112	Goddard
9	peak picking	Sparky	3.112	Goddard
10	geometry optimization	TALOS+		Shen, Cornilescu, Delaglio and Bax
11	dihedral angle constraints	TALOS+		Shen, Cornilescu, Delaglio and Bax
12	structure quality analysis	PSVS	1.4	Bhattacharya and Montelione
13	structure quality analysis	MolProbity	3.18	Richardson
14	pdb coordinate analysis	PdbStat	5.4	Tejero & Montelione

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.

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2LJW

Solution NMR structure of Alr2454 protein from Nostoc sp. strain PCC 7120, Northeast Structural Genomics Consortium Target NsR264