2K53

NMR solution structure of A3DK08 protein from Clostridium thermocellum: Northeast Structural Genomics Consortium Target CmR9

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
2	2D 1H-15N HSQC	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
3	2D 1H-13C HSQC	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
4	2D 1H-13C HSQC	1.05 mM [U-10% 13C; U-99% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
5	3D CBCA(CO)NH	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
6	3D HNCACB	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
7	3D HNCO	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
8	3D HBHA(CO)NH	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
9	3D HCCH-TOCSY	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
10	3D 1H-13C NOESY	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
11	3D 1H-15N NOESY	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 Protein	90% H2O/10% D2O	5mM CaCl2, 100mM NaCl	6.5	1 atm	293
12	3D 1H-13C NOESY	0.91 mM [U-100% 13C; U-100% 15N] A3DK08 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	800

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 the sidechain assignments were completed manually. Automated NOESY assignments were made using AUTOSTRUCTURE and CYANA-2.1. Dihedral angle constraints were obtained from TALOS. The structure calculations were done excluding the six HIS from the 8-residue C-terminal tag(LEHHHHHH). Completeness of assignments excluding the HHHHHH: Backbone:100% Sidechain(aliphatic): 99% Sidechain (Aromatic): 90% Stereospecific Methyl assignments:100%. The assignments were validated using AVS software. Final structure quality factors(excluding the HHHHHH) determined using PSVS-v1.3: Ordered residues are defined as residues 2-28,33-34,37-69.(a) RMSD (ordered residues)all backbone atoms:0.4A, all heavy atoms: 0.7A; RMSD (all residues) backbone: 0.7A and heavy atoms: 1.0A. (b) Ramachandran statistics for ordered residues : Most favoured regions: 97.1%, additionally allowed regions: 2.9%.(C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI,0.21/1.14, ALL,0.24/1.42 (D) MOLPROBITY CLASH SCORE (RAW/Z-): 17.26/-1.44. (E) RPF scores for the goodness of the fit to NOESY data: Recall:0.984, PRECISION: 0.848, F-measure: 0.911 and final DP-score:0.743. (f) Number of close contacts for 20 models: 6. RMS deviation for bond angles:0.6deg. RMS deviation for bond lengths 0.008A.	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)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinement	CNS	2.0.6	Brunger, Adams, Clore, Gros, Nilges and Read
2	structure solution	CYANA	2.1	Guntert, Mumenthaler and Wuthrich
3	chemical shift assignment	AutoAssign	2.4.0	Zimmerman, Moseley, Kulikowski and Montelione
4	noesy assignments	AutoStructure	2.2.1	Huang, Tejero, Powers and 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.