2L3F
Solution NMR Structure of a putative Uracil DNA glycosylase from Methanosarcina acetivorans, Northeast Structural Genomics Consortium Target MvR76
SOLUTION NMR
NMR Experiment | ||||||||
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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] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
2 | 2D 1H-13C HSQC | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
3 | 3D 1H-15N NOESY | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
4 | 3D 1H-13C NOESY aliphatic | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
5 | 3D 1H-13C NOESY aromatic | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
6 | 2D 1H-13C HSQC high resolution (L/V methyl stereoassignment) | 1.15 mM [U-5% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
7 | 3D HNCO | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
8 | 3D HN(CA)CO | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
9 | 3D HNCA | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
10 | 3D HN(CO)CA | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
11 | 3D CBCA(CO)NH | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
12 | 3D HNCACB | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
13 | 3D HBHA(CO)NH | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
14 | 3D HCCH-TOCSY | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
15 | 3D CCH-TOCSY | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
16 | 3D HCCH-COSY | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
17 | 3D HNHA | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
18 | 3D HBHANH | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
19 | 1D 15N T1 and T2 | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
20 | 2D 1H-15N hetNOE | 0.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
21 | NH J-modulation | 0.80 mM [U-5% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 4 % C12E5 PEG | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
22 | NH J-modulation | 1.15 mM [U-5% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 7 % Polyacrylamide gel | 90% H2O/10% D2O | 6.5 | ambient | 298 |
NMR Spectrometer Information | |||
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Spectrometer | Manufacturer | Model | Field Strength |
1 | Bruker | AVANCE | 800 |
2 | Bruker | AVANCE | 600 |
3 | Varian | INOVA | 600 |
NMR Refinement | ||
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Method | Details | Software |
simulated annealing | THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2729 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 234 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (18.4 CONSTRAINTS PER RESIDUE, 5.4 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 161 BY PSVS 1.4). IN ADDITION, 210 RESOLVED N-H RESIDUAL DIPOLAR COUPLINGS FOR ORDERED RESIDUES OBTAINED FROM TWO ALIGNMENTS WERE INCLUDED IN ALL STRUCTURE CALCULATIONS. 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 | |
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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 | |
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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 CROYOPROBE. 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 His5): BACKBONE, 98.5%, SIDE CHAIN, 91.0%, AROMATICS, 84.3%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 64.0%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 161, PSVS 1.4), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 2-34,39-81,92-144,146-160: (A) RMSD (ORDERED RESIDUES): BB, 0.6, HEAVY ATOM, 1.1. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 89.1%, ADDITIONALLY ALLOWED, 10.8%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.26/-0.71, ALL, -0.15/-0.89. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 16.95/-1.38 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1 TO 161): RECALL, 0.951, PRECISION, 0.899, F-MEASURE, 0.924, DP-SCORE, 0.722. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 11. THE FINAL FIVE 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 | ||||
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# | Classification | Version | Software Name | Author |
1 | refinement | CNS | 1.1 | Brunger, Adams, Clore, Gros, Nilges and Read |
2 | refinement | CYANA | 3.0 | Guntert, Mumenthaler and Wuthrich |
3 | structure solution | CYANA | 3.0 | Guntert, Mumenthaler and Wuthrich |
4 | rpf analysis | AutoStructure | 2.2.1 | Huang, Tejero, Powers and Montelione |
5 | structure quality analysis | PSVS | 1.4 | Bhattacharya and Montelione |
6 | processing | NMRPipe | 2.3 | Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax |
7 | structure quality analysis | MolProbity | 3.18 | Richardson |
8 | collection | TopSpin | 2.1 | Bruker Biospin |
9 | data analysis | TopSpin | 2.1 | Bruker Biospin |
10 | pdb coordinate analysis | PdbStat | 5.1 | Tejero & Montelione |
11 | chemical shift assignment | PINE | 1.0 | Bahrami, Markley, Assadi, and Eghbalnia |
12 | data analysis | Sparky | 3.112 | Goddard |
13 | peak picking | Sparky | 3.112 | Goddard |
14 | geometry optimization | TALOS+ | Shen, Cornilescu, Delaglio and Bax | |
15 | rdc analysis | PALES | PALES (Zweckstetter, Bax) | |
16 | collection | VnmrJ | 2.1 | Varian |
17 | data analysis | VnmrJ | 2.1 | Varian |