2K1G

Solution NMR structure of lipoprotein spr from Escherichia coli K12. Northeast Structural Genomics target ER541-37-162

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
2	2D 1H-13C HSQC	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
3	3D HNCO	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
4	3D HN(CA)CO	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
5	3D HN(COCA)CB	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
6	3D HNCACB	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
7	3D HBHA(CO)NH	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
8	3D HCCH-COSY	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
9	3D simultaneous CN NOESY	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
10	3D CCH-TOCSY aromatic	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
11	3D 1H-13C NOESY aromatic	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	100% D2O	0.1	6.5	ambient	298
12	3D simultaneous CN NOESY	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	100% D2O	0.1	6.5	ambient	298
13	3D CCcoNH TOCSY	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
14	3D HCCH-TOCSY	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
15	3D CCH-TOCSY aliphatic	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
16	3D HNHA	1.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
17	2D 1H-13C high res. (L/V stereo assignment)	1.03 mM [U-5% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298
18	2D 1H-15N hetNOE	1.03 mM [U-5% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide	95% H2O/5% D2O	0.1	6.5	ambient	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	Avance	800
2	Varian	INOVA	600

NMR Refinement
Method	Details	Software
simulated annealing	THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2525 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 138 DIHEDRAL ANGLE CONSTRAINTS, AND 48 HYDROGEN BOND CONSTRAINTS (21.0 CONSTRAINTS PER RESIDUE, 7.1 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 37 TO 162 BY PSVS 1.3). STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 2.1. 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, AND USING NEUTRAL HISTIDINES AT POSITIONS 119 and 131.	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 AND STATIC LIGHT SCATTERING. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. AUTOMATED BACKBONE ASSIGNMENTS WERE MADE USING AUTOASSIGN, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 2.1. DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOS. HYDROGEN BOND CONSTRAINTS WERE DETERMINED USING BOTH AUTOSTRUCTURE AND CYANA, AND WERE APPLIED ONLY IN THE FINAL REFINEMENT STAGE (CNS) OF THE STRUCTURE DETERMINATION. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 97.9%, SIDE CHAIN, 94.2%, AROMATICS, 91.8%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 83.3%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 37 TO 162, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 38-58,61-93,96-113,118-124,128-159: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 1.0. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 88.4%, ADDITIONALLY ALLOWED, 11.5%, GENEROUSLY ALLOWED, 0.1%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.46/-1.49, ALL, -0.27/-1.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 18.98/-1.73. (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 37-162): RECALL, 0.985, PRECISION, 0.925, F-MEASURE, 0.954, DP-SCORE, 0.816. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 8. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION. COORDINATES FOR THE FOLLOWING RESIDUES ARE NOT WELL DETERMINED [S(PHI) + S(PSI) < 1.8]: 37,59-60,94-95,114-117,125-127,160-162.

Additional NMR Experimental Information

Details

THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY AND STATIC LIGHT SCATTERING. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. AUTOMATED BACKBONE ASSIGNMENTS WERE MADE USING AUTOASSIGN, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 2.1. DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOS. HYDROGEN BOND CONSTRAINTS WERE DETERMINED USING BOTH AUTOSTRUCTURE AND CYANA, AND WERE APPLIED ONLY IN THE FINAL REFINEMENT STAGE (CNS) OF THE STRUCTURE DETERMINATION. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 97.9%, SIDE CHAIN, 94.2%, AROMATICS, 91.8%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 83.3%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 37 TO 162, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 38-58,61-93,96-113,118-124,128-159: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 1.0. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 88.4%, ADDITIONALLY ALLOWED, 11.5%, GENEROUSLY ALLOWED, 0.1%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.46/-1.49, ALL, -0.27/-1.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 18.98/-1.73. (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 37-162): RECALL, 0.985, PRECISION, 0.925, F-MEASURE, 0.954, DP-SCORE, 0.816. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 8. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION. COORDINATES FOR THE FOLLOWING RESIDUES ARE NOT WELL DETERMINED [S(PHI) + S(PSI) < 1.8]: 37,59-60,94-95,114-117,125-127,160-162.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	collection	TOPSPIN	1.3	Bruker Biospin
2	collection	VNMR	6.1C	Varian
3	chemical shift assignment	AutoAssign	2.4.0	Zimmerman, Moseley, Kulikowski and Montelione
4	processing	NMRPipe	2.3	Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
5	peak picking	SPARKY	3.110	Goddard
6	data analysis	SPARKY	3.110	Goddard
7	structure solution	CYANA	2.1	Guntert, Mumenthaler and Wuthrich
8	refinement	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
9	structure solution	CNS	1.2	Brunger, Adams, Clore, Gros, Nilges and Read
10	rpf validation	AutoStructure	2.2.1	Huang, Tejero, Powers and Montelione
11	structure validation	PSVS	1.3	Bhattacharya and Montelione
12	pdb analysis	PdbStat	5.0	Tejero and Montelione

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.

Prepare Data

Validate Data

Deposit Data

Help and Resources

2K1G

Solution NMR structure of lipoprotein spr from Escherichia coli K12. Northeast Structural Genomics target ER541-37-162