TABLE 5.

Troubleshooting table.

Step	Problem	Possible reason	Solution
8	Δ R2 equal to 0 at all residue positions; DEST is unlikely to be observed if Δ R2 is not substantially greater than 0.2 s−1	Insufficient population of dark state	Modify sample conditions or concentrations to increase the conversion rate from the NMR-visible to the dark species. This can often be accomplished by increasing the concentration of the dark state
	Negative values of Δ R2 are computed or R2 is not repeatable	Imperfect reference and dark-state sample condition matching. Small changes in pH (on the order of 0.2 pH units) near physiological pH of 7 can change the rate of 1HN exchange with 1H from bulk water on the order of 1 s−1 or greater	Option 1: the reference sample should be changed (i.e., pH adjusted) such that the sample conditions match. Resonance positions of 1H-15N correlations of histidine residues can be used as sensitive internal reporters of the relative pH of the two samples, and titrating the pH of the reference sample to precisely overlay with the 1H-15N correlation spectrum is a useful way to ensure sample consistency. If pH adjustment after sample creation is not possible, several reference samples with slightly different sample pH can be screened for the best overlay and/or used for R2 measurement. Option 2: pulse sequences that remove the contribution of water exchange to the measured relaxation rates12 can be used in place of standard 15N-R2 measurements; or lower the sample temperature
10	No DEST (off-resonance) saturation is observed	Saturation power is too weak	Increase the saturation power (Box 1)
		$R_2^{\text{dark}}$ is too small to give rise to DEST	Increase $R_2^{\text{dark}}$ by decreasing temperature or increasing the size of the dark state
16	Warning concerning local minima of optimization	It is difficult to guarantee a global optimum solution in an extremely large parameter space search	Convergence may be confirmed by independent fitting with different initial parameters, leading to the same solutions
17	Poor fit to DEST and/or ΔR2 data	Model is inappropriate for the system of interest	Change the fit type of the model (e.g., change from fit type 2, two-state model, to fit type 3 or 1, three-state or tethered/ bound models, respectively)