, where V is the box volume, NA is Avogadro's number, pB(V) is the bound probability, v is the interacting volume (the volume where protein and ligand interact but the ligand is not in the bound pose), and pv is the probability of finding the ligand within the volume V. In the case of ligand binding with an IDP, the difference between the interacting and bound volumes is subtle (with no precise binding pose). By defining the bound state considering the configurations where the minimum distance between Aβ42 and 10074-G5 is less than 0.33 nm and as interacting the state where the minimum distance is less than 0.4 nm, a dissociation constant of 683±7 µM was calculated for multi-eGO (with the error calculated as standard deviation of the mean of 4 replicates of 2 µs each) (see Figure ), in comparison with 381±5 µM obtained by analyzing the training simulation (where the error is calculated using a bootstrapping method with 95th percentile). To note, the difference between the training and multi-eGO dissociation constants corresponds to 1.5 kJ/mol in free energy, less than the thermal fluctuation energy (~2.5 kJ/mol at 300 K). However, these values are not in quantitative agreement with the experimental value of 7-40 µM, most likely because of limitations in force-field accuracy. In this case, a substantial advantage of using multi-eGO is represented by the possibility of updating the model to reproduce the experimental affinity by changing a single parameter, 𝜀 ' , which represents the energy scale for the Aβ42:10074-G5 interaction, without the requirement to repeat the computationally intensive all-atom training simulation. A 𝜀 ' of 0.46 kJ/mol corresponded to a dissociation constant of 18±1 µM (Figure ), before and after volume correction, where the data represent the mean, and the standard deviation of the mean obtained from 5 replicates of 2 µs each. In silico titration simulation of the Aβ42:10074-G5 multi-eGO model. The updated model was then used to study Aβ42:10074-G5 binding in the presence of an increasing number of 10074-G5 molecules. Following in vitro experiments, we performed an in-silico titration experiment mixing 1 µM Aβ42 (consisting of 10 monomers in a 255 nm cubic box, cf. Fig. ) with increasing concentrations of 10074-G5, namely 1:1, 1:6, 1:10, 1:20, 1:40 and 1:80 Aβ42:10074-G5 ratios. At each ratio, 5 µs long multi-eGO simulation were run (see Fig. for binding probability as a function of time and block averaging), and the concentration of bound Aβ42:10074-G5 was fitted as a function of ligand concentration using the equation [𝐿𝑃] =