Fig 1.
Schematic outline of the fitting protocol adopted by decon1d.
Fig 2.
Fitting of simulated spectra with decon1d.
a-d) Non-exchange broadened spectra of varying signal-to-noise ratio and number, width, frequency and height of component peaks were simulated (top row). decon1d was then used to fit these simulated spectra allowing for either fixed phase (middle row) or variable phase (bottom row). The color of the component peaks identified in each fit serves as a visual aid for comparisons between fits as it identifies the approximate chemical shift of the peak center, indicated by the colored bar on the bottom. The difference between the data and the fit (residual error) is shown in grey and the sum of individual fitted peaks is shown in green. An alternate deconvolution of the variable phase fit for column c is displayed in S5 Fig.
Fig 3.
Lower signal-to-noise ratio leads to decreased peak assignment.
a-e) Fits of simulated data with signal-to-noise ratio: a) 5 b) 10, c) 25, d) 75 and e) 244. f) Input simulated NMR spectra showing the true underlying peaks that make up the spectra. Signal-to-noise was calculated from the highest signal value divided by the root mean square value of the noise in a region devoid of signal. An alternate fit of the lowest signal to noise data (panel a) was found with a BIC value 4.67 higher than the model shown, with the only substantial difference being that the prediction of rightmost peak chemical shift is -3.58 ppm (not shown) rather than -3.12 ppm (shown).
Fig 4.
Out-of-phase data are well fit by decon1d.
Simulated data (input) were fit with decon1d allowing the phase to vary (model). a) The input and fit models are nearly identical for these incorrectly phased simulated spectra. b) The fractional population, center, full width at half maximum peak height (FWHM) and phase of the simulated spectrum (dashed and solid lines) and the fits (colored dots) were graphed as a function of the phase of the simulated spectrum (x-axis). In general these fits are not adversely affected by poor phasing. The difference between the data and the fit (residual error) is shown in grey and the sum of individual fitted peaks is shown in green.
Fig 5.
Intermediate exchange data are well fit by decon1d.
Data were simulated using LineShapeKin and fit using decon1d. a) The spectrum from a single nucleus exchanging between two chemical shift environments was simulated with near equal populations (left panel; 48%:52%) and skewed populations (right panel; 25%:75%) at varying exchange rate to chemical shift difference values (kex/Δδ, displayed numbers) and the best model of the component spectral lines was determined by decon1d. Vertical gray dashed lines indicate the true chemical shifts in the absence of exchange. b) Fitted parameters from the models in panel a. c) Simulated spectra from a single nucleus exchanging between four chemical shift environments with similar populations at varying kex/Δδ values (displayed numbers) and the best model as determined by decon1d. The difference between the data and the fit (residual error) is shown in grey and the sum of individual fitted peaks is shown in green.
Fig 6.
Fit of real experimental data.
The ligand binding domain of PPARγ C285S/K474C was treated with BTFA and then NMR was performed at 298K. Deconvolution of the 19F NMR signal was carried out using the indicated programs. In each case the difference between the data and the fit (residual error) is shown in grey and the sum of individual fitted peaks is shown in green.
Fig 7.
The integral of the 19F NMR signal was calculated to the left of the dotted red vertical lines in Fig 6 (from -83 ppm to -84.05 ppm, left, circles) and to the right of the dotted red vertical lines in Fig 6 (from -84.05 to -85 ppm, right, boxes) in two independent experiments (blue and black) and plotted as a function of total delay time in the 19F T2 experiment described in the text. Two near-zero data points from repeat two at 0.3s delay are not shown for clarity (-0.012 and 0.023 for left and right respectively) but are included in the fits. Spectra used in the 19F T2 calculation are shown in supporting information (S10 Fig).
Table 1.
Comparison of experimental to fit parameters.