Distortion of a ligand during umbrella sampling simulations is a concern in PMF calculations, especially for flexible peptides, which may lead to erroneous results if the distortion becomes permanent after the ligand. We have checked against this possibility by calculating the average rmsd of m -GIIIA in each umbrella sampling window. As shown in Povidone iodine Figure 5, the toxin undergoes some distortion while it is pulled out of the binding pocket but the elastic energy associated with this distortion is recovered once the toxin is in the bulk region as indicated by the return of the rmsd to the bulk value. Lack of distortion is also verified by aligning the m-GIIIA structures from the last umbrella window with the NMR structure. Two main questions in PMF Mercaptopurine (6-MP) calculations are how far the PMF should be extended and how long each window should be run. The first is addressed by appearance of a flat region in the PMF which indicates that the ligand has reached the bulk region. The second question is rarely addressed in PMF calculations but it is equally important because without sufficient data, one is likely to obtain a wrong answer. We address this issue by performing block data analysis of the PMF data. That is, we construct PMFs from 2 ns blocks of data and slide the blocks in 1 ns steps over the range of the available data. As shown in Figure 6, the PMFs initially drop monotonically and then fluctuate around a base line. During the first phase, the PMFs drop because of the improved screening of the channel�Ctoxin interactions as the system equilibrates. In the second phase, fluctuations of the PMFs around a base line are of statistical nature and indicate that the system has been equilibrated. A common practice in PMF calculations is to exclude an arbitrary amount of data for equilibration, and consider the rest of the data in production of the PMF. As seen from the convergence study in Figure 6, this could result in mixing of the equilibration and production data, which would lead to an overestimation of the binding affinity. The final PMF for the NaV1.4�C m -GIIIA complex is indicated by a thick black line in Figure 6.