2K5Q

NMR Solution structure of membrane associated protein from Bacillus cereus: Northeast Structural Genomics Consortium Target: BcR97A

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
2	2D 1H-13C HSQC	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
3	3D HNCO	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
4	3D CBCA(CO)NH	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
5	3D HNCACB	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
6	3D HBHA(CO)NH	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
7	3D C(CO)NH	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
8	3D HCCH-TOCSY	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
9	3D HCCH-COSY	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
10	3D 13C-edited_NOESY	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
11	3D 1H-15N NOESY	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
12	3D 13C-edited_NOESY	1.25 mM [U-100% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
13	2D 1H-15N HSQC	0.77 mM [U-10% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
14	2D 1H-13C HSQC	0.77 mM [U-10% 13C; U-100% 15N] BcR97A protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293

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

NMR Refinement
Method	Details	Software
simulated annealing	The structure was determined using triple resonance NMR spectroscopy. Automated backbone resonance assignments were made using AutoAssign and pattern picker algorithms developed for automated assignments of GFT NMR data. Side chain assignments were completed manually. Automated NOESY assignments were made using AUTOSTRUCTURE and structure solution was determined using AUTOSTRUCTURE and CYANA-2.1. 176 structures were calculated and 20 best conformers were then refined in a shell of water using CNS. Initial Dihedral constriants were obtained from TALOS. The structure calculations were done including the C-terminal tag LEHHHHHH. Completeness of assignments excluding the 8-residue tag are: Backbone ~98.5%, sidechain ~ 95%, stereospecific methyl assignments 100%. The assignments were validated using the AVS software. Final structure quality factors determined using PSVS software: Ordered residues are defined as: 10-22,32-42,47-58,63-69,76-93. (a) RMSD(ordered residues) all Backbone atoms 0.9A; all heavy atoms 1.4A. (b) Ramachandran statistics for all ordered residues: Most favoured regions:85.9%, Additionally allowed regions 14.0%, Generously allowed region:0.2%. (c) Procheck scores for ordered residues (Raw/Z)phi-psi -0.73/-2.56, All:-0.49/-2.90 (d) MolProbity clash score (Raw/Z): 17.11/-1.41. (e) RPFscores for goodness of fit to NOESY data : Recall:0.963 Precision: 0.805F-measure:0.877 DP-score: 0.848	AutoAssign

NMR Ensemble Information
Conformer Selection Criteria	structures with the lowest energy
Conformers Calculated Total Number	176
Conformers Submitted Total Number	20
Representative Model	1 (lowest energy)

Additional NMR Experimental Information
Details	GFT (4,3)D data was acquired for backbone assignments.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	chemical shift assignment	AutoAssign	2.2.1	Zimmerman, Moseley, Kulikowski and Montelione
2	chemical shift assignment	AutoStructure	2.2.1	Huang, Tejero, Powers and Montelione
3	structure solution	AutoStructure	2.2.1	Huang, Tejero, Powers and Montelione
4	structure solution	CYANA	2.1	Guntert, Mumenthaler and Wuthrich
5	geometry optimization	CYANA	2.1	Guntert, Mumenthaler and Wuthrich
6	refinement	CNS	2.0.6	Brunger, Adams, Clore, Gros, Nilges and Read
7	geometry optimization	CNS	2.0.6	Brunger, Adams, Clore, Gros, Nilges and Read

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.