# Fast and Interactive Positioning of Proteins within Membranes
## Appendix A  
Here, we explain the uncertainties that exist in our implementation of the IMPALA method due to missing information from the literature, and how we attempted to determine the most likely parameter combination to reproduce the original calculations. One of the causes of the discrepancy is in the actual sampling. In addition to our exhaustive parameter grid search, we implemented an experimental simple Monte Carlo approach to mimic [PERSON]'s sampling approach. However, insufficient details were reported on the exact implementation of the Monte Carlo moves, so we had to make assumptions about the translation and rotation conditions. The reference work mentions rotation, but it is unclear whether only a single angle was considered or whether both the insertion angle and the roll angle were sampled separately. Our test results mainly confirmed that the sampling approach had an impact on the results and showed significant variability between 10 runs, while [PERSON] only performed two replicates.  
The equations used in the early publications of the IMPALA method vary without any justification. In particular, the definition of **C(z)** in one publication was given as water content at a certain level and ranged from 0 to 1, whereas in another publication, it was shifted to the range \(-0.5\) to \(+0.5\). We tested both definitions, which are related by a simple shift in the resulting energies, but ultimately used the latter definition. The reason is that the alternative implementation did not make sense from a physicochemical point of view, as it implied that the positioning of the protein always resulted in an energy penalty and never in a favorable energetic contribution. A similar problem was observed for the \(\text{a}_{\text{ip}}\) parameter, which was reported with a positive or negative sign depending on the publication, and its units are never explicitly indicated. The addition of hydrogens is not explicitly mentioned: in early work on peptides, hydrogens are clearly present. In later work on membrane proteins, the underlying PDB structures do not contain hydrogen atoms, and it is not known whether or how the hydrogen atoms were eventually added. If they had been omitted, the calibration of the per-atom transfer energy contributions would need to be adjusted, but this has not been reported. It seems likely, therefore, that the hydrogen atoms were added. If so, the parameters chosen for the addition of hydrogen could affect the results. We have already mentioned the exact atomic radii as important parameters for the calculation and took these parameters from one of the original articles. These parameters are important for the calculation of SASA, which in turn depends on the exact algorithm and parameters such as resolution, which we discuss in more detail in Section 2.3.3.  
A final element concerns the measurement of insertion depth and angle. The depth, measured as the position of the center of mass, leaves little doubt. However, as far as the definition and measurement of the angle are concerned, as already mentioned in the manuscript, we found that there are incoherences between text and illustrations in the original works on OmpA, which means that we are not 100% sure to measure exactly the same angle as it is given in the literature.  
In addition to the experiments with OmpA, a complete protein, we attempted to reproduce previous work with the simpler peptide melittin described in [18]. An alpha-helical model was used and the hydrogens are clearly present. To reproduce this experience, we used our experimental Monte Carlo implementation. Figure 11 shows the comparison between the originally reported results and our experiments for the insertion angle. It can be seen that several important features of the graph are well reproduced, particularly three minima and a saddle point. The good agreement between our experiment and the reference implementation emphasizes that the results are well reproduced if the uncertainties can be removed.