2KPN

Solution NMR structure of a Bacterial Ig-like (Big_3) domain from Bacillus cereus. Northeast Structural Genomics Consortium target BcR147A

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
2	2D 1H-13C HSQC	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
3	3D 1H-15N NOESY	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
4	3D 1H-13C NOESY aliphatic	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
5	3D 1H-13C NOESY aromatic	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
6	2D 1H-13C HSQC high resolution (L/V methyl stereoassignment)	0.78 mM [U-5% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
7	2D 1H-15N hetNOE	0.78 mM [U-5% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
8	3D HNCO	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
9	3D HN(CA)CO	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
10	3D HNCA	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
11	3D CBCA(CO)NH	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
12	3D HNCACB	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
13	3D HBHA(CO)NH	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
14	3D HCCH-TOCSY	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
15	3D HCCH-COSY	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
16	3D CCH-TOCSY	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	ambient	298
17	1D 1H-15N T1 and T2	0.94 mM [U-100% 13C; U-100% 15N] BcR147A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS	90% H2O/10% D2O	0.2	6.5	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 1806 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 132 DIHEDRAL ANGLE CONSTRAINTS, AND 9 METAL-OXYGEN DISTANCE CONSTRAINTS (20.8 CONSTRAINTS PER RESIDUE, 7.3 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 97 BY PSVS 1.3). 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.	TOPSPIN

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 HHHHHH): BACKBONE, 97.5%, SIDE CHAIN, 99.2%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 90%, STEREOSPECIFIC SIDE CHAIN NH2: 66.7%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUE NUMBERS 1 TO 97, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 7-85: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 0.8. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 96.3%, ADDITIONALLY ALLOWED, 3.7%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.45/-1.46, ALL, -0.27/-1.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 14.17/-0.91 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-97): RECALL, 0.979, PRECISION, 0.939, F-MEASURE, 0.959, DP-SCORE, 0.854. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 3. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION. THE LIGANDS INVOLVED IN THE CALCIUM ION COORDINATION SPHERE ARE CONSISTENT WITH CHANGES IN THE NH-HSQC SPECTRUM UPON REMOVAL/ADDITION OF CA2+.

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 HHHHHH): BACKBONE, 97.5%, SIDE CHAIN, 99.2%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 90%, STEREOSPECIFIC SIDE CHAIN NH2: 66.7%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUE NUMBERS 1 TO 97, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 7-85: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 0.8. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 96.3%, ADDITIONALLY ALLOWED, 3.7%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.45/-1.46, ALL, -0.27/-1.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 14.17/-0.91 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-97): RECALL, 0.979, PRECISION, 0.939, F-MEASURE, 0.959, DP-SCORE, 0.854. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 3. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION. THE LIGANDS INVOLVED IN THE CALCIUM ION COORDINATION SPHERE ARE CONSISTENT WITH CHANGES IN THE NH-HSQC SPECTRUM UPON REMOVAL/ADDITION OF CA2+.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	collection	TOPSPIN	2.1	Bruker Biospin
2	data analysis	TOPSPIN	2.1	Bruker Biospin
3	processing	NMRPipe	2.3	Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
4	data analysis	SPARKY	3.112	Goddard
5	peak picking	SPARKY	3.112	Goddard
6	chemical shift assignment	PINE	1.0	Bahrami, Markley, Assadi, and Eghbalnia
7	structure solution	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
8	refinement	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
9	rpf analysis	AutoStructure	2.2.1	Huang, Tejero, Powers and Montelione
10	structure quality analysis	PSVS	1.3	Bhattacharya and Montelione
11	structure quality analysis	MolProbity	3.15	Richardson
12	pdb coordinate analysis	PdbStat	5.1	Tejero and Montelione
13	dihedral angle constraints	TALOS	plus	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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2KPN

Solution NMR structure of a Bacterial Ig-like (Big_3) domain from Bacillus cereus. Northeast Structural Genomics Consortium target BcR147A