2KWB
Minimal Constraint Solution NMR Structure of Translationally-controlled tumor protein (TCTP) from C.elegans, Northeast Structural Genomics Consortium Target WR73
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 2D 1H-15N HSQC | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 2 | 2D 1H-15N TROSY-HSQC | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 3 | 2D 1H-13C HSQC aliphatic | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 4 | 3D HNCO | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 5 | 3D HN(CA)CO | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 6 | 3D TROSY-HNCACB | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 7 | 3D TROSY-HN(CO)CACB | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 8 | 3D 1H-15N NOESY | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 9 | 3D 1H-13C NOESY aliphatic | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 10 | 3D 15N-15N-1H NOESY | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 11 | 3D 13C-13C-1H NOESY | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 12 | 3D 15N-13C-1H NOESY | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 13 | 3D 13C-15N-1H NOESY | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 14 | 3D C(CO)NH TOCSY | 0.82 mM [U-13C; U-15N; U-2H; ILVFY-1H] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 6.5 | ambient | 298 | ||
| 15 | 2D 1H-13C HSQC high resolution | 0.85 mM [U-5% 13C; U-100% 15N] WR73, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 50 uM DSS, 0.02 % sodium azide | 90% H2O/10% D2O | 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 MINIMAL CONSTRAINT STRUCTURES ARE BASED ON A TOTAL OF 976 CONFORMATIONALLY-RESTRICTING DISTANCE CONSTRAINTS, 272 DIHEDRAL ANGLE CONSTRAINTS, AND 126 HYDROGEN BOND CONSTRAINTS (7.6 CONSTRAINTS PER RESIDUE, 2.3 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 183 BY PSVS 1.4). 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 | |
|---|---|
| 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 AT 298 K BY 15N T1/T2 RELAXATION AND STATIC LIGHT SCATTERING. THE STRUCTURE IS A MINIMAL CONSTRAINT STRUCTURE DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. ALL NOESY DATA WERE ACQUIRED AT 800 MHZ USING A 5-MM CRYOPROBE. BACKBONE ASSIGNMENTS WERE MADE USING PINE, AND THE SIDE CHAIN METHYL ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 3.0. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOSplus. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUE NUMBERS 1 TO 183, PSVS 1.4), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 2-12,14-16,18-38,66-106,111-146,152-181: (A) RMSD (ORDERED RESIDUES): BB, 1.0, HEAVY ATOM, 1.5. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 89.0%, ADDITIONALLY ALLOWED, 10.9%, GENEROUSLY ALLOWED, 0.1%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.34/-1.02, ALL, -0.31/-1.83. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 20.78/-2.04 (E) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 55. 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]: 1,13,17,39-65,107-110,147-151,182-183. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | refinement | CNS | 1.2 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 2 | structure solution | CNS | 1.2 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 3 | geometry optimization | CNS | 1.2 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 4 | structure solution | CYANA | 3.0 | Guntert, Mumenthaler and Wuthrich |
| 5 | chemical shift assignment | PINE | 1.0 | Bahrami, Markley, Assadi, and Eghbalnia |
| 6 | processing | NMRPipe | 2.3 | Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax |
| 7 | data analysis | Sparky | 3 | Goddard |
| 8 | peak picking | Sparky | 3 | Goddard |
| 9 | chemical shift assignment | Sparky | 3 | Goddard |
| 10 | collection | TopSpin | 2.1 | Bruker Biospin |
| 11 | collection | VnmrJ | Varian | |
| 12 | structure validation | PSVS | 1.4 | Bhattacharya and Montelione |
| 13 | dihedral angle constraints | TALOS | plus | Cornilescu, Delaglio and Bax |
| 14 | pdb coordinate analysis | PdbStat | 5.1 | Tejero and Montelione |
