2G5M

Spinophilin PDZ domain


SOLUTION NMR
NMR Experiment
ExperimentTypeSample ContentsSolventIonic StrengthpHPressureTemperature (K)Spectrometer
13D 15N resolved [1H,1H] NOESY1.8 mM PDZ domain, 15N samples; 20 mM Na phosphate buffer 50 mM NaCl, pH 6.5; 90% H2O, 10% D2O90% H2O/10% D2O20 mM Na phosphate; 50 mM NaCl6.5ambient298
23D 13C resolved [1H,1H] NOESY1.8 mM PDZ domain, 13C/15N samples; 20 mM Na phosphate buffer 50 mM NaCl, pH 6.5; 90% H2O, 10% D2O90% H2O/10% D2O20 mM Na phosphate; 50 mM NaCl6.5ambient298
32D 1H-1H NOESY1.8 mM PDZ domain, 13C/15N samples; 20 mM Na phosphate buffer 50 mM NaCl, pH 6.5; 90% H2O, 10% D2O100% d2020 mM Na phosphate; 50 mM NaCl6.5ambient298
NMR Spectrometer Information
SpectrometerManufacturerModelField Strength
1BrukerAVANCE500
NMR Refinement
MethodDetailsSoftware
simulated annealing torsion angle dynamicsThe amino acid sequence, the chemical shift assignment, and the NOESY spectra were input for the automated NOESY peak picking and NOE assignment method of ATNOS/CANDID/CYANA. The results of ATNOS/CANDID/CYANA were refined by manual peak adjustment and additional calculations in CYANA. The 20 conformers from the ATNOS/CANDID cycle 7 with the lowest residual CYANA target function values were energy-minimized in a water shell with the program CNS.TopSpin
NMR Ensemble Information
Conformer Selection Criteriastructures with the lowest energy
Conformers Calculated Total Number50
Conformers Submitted Total Number20
Representative Model1 (closest to the average)
Additional NMR Experimental Information
DetailsThe following spectra were used to achieve the sequence-specific backbone and side chain assignments of all aliphatic residues: 2D [1H,15N]-HSQC, 2D [1H,13C]-HSQC, 3D HNCACB, 3D CBCA(CO)NH, 3D CC(CO)NH, 3D HNCO, 3D HNCA, 3D HBHA(CO)NH, 3D 15N-resolved [1H,1H]-TOCSY, 3D HC(C)H-TOCSY6. The 2D [1H,1H]-NOESY, 2D [1H,1H]-TOCSY and 2D [1H,1H]-COSY spectra of the Spinophilin493-602 sample in D2O solution after complete H/D exchange of the labile protons were used for the assignment of the aromatic side chains. The NMR spectra were processed with Topspin1.3, and analyzed with the CARA software package. Spectra used for the structure calculation were: 3D 15N-resolved [1H,1H]-NOESY, 3D 13C-resolved [1H,1H]-NOESY (mixing time of 85 ms) and 2D [1H,1H]-NOESY (mixing time 85 ms, D2O solution).
Computation: NMR Software
#ClassificationVersionSoftware NameAuthor
1processingTopSpin1.3Bruker
2structure solutionCYANA2.0peter guntert
3refinementCNS1.1Brunger
4structure solutionatnos/candid2.0Torsten Herrmann
5data analysisCARA1.5Rochus Keller