To test this hypothesis, a dilution series was set up, ranging from 500 nM to 10 nM protein concentration, with the CuNTA concentration in each sample calculated to yield approximately 90% binding (Table ). Standard constant-time (ct) RIDME measurements were performed with identical experimental parameters for each sample, with the exception of the averaging times (and thus, the number of scans) varying between 2 hours for the 500 nM sample to approximately 48 hours for the 10 nM sample (Figure , Figure ). ctRIDME data were processed using a Tikhonov Regularisation procedure within DeerAnalysis2022 61 as described previously, and the obtained distance distributions for all samples were in excellent agreement with the simulated distribution. It should be noted that our in-house RIDME processing protocol follows a very conservative approach, potentially overestimating the uncertainty in the resulting distance distribution (for details see SI). While we obtained high confidence distance distributions down to 50 nM protein, lower concentrations led to significantly increased uncertainties including broader confidence bands and such data should only be interpreted with great care. Thus, concentrations below 50 nM remain a challenge for ctRIDME even when using CuNTA-SLIM label pairs, and higher-quality data with lower uncertainties than seen for 10 and 25 nM will be required to e.g., determine whether multiple distance populations are present, as observed previously. This is in line with data obtained from alternative deep neural network processing (DEERNet 62 with RIDME background model