Please don’t hesitate to ask! You can reach us by email: [email protected].

First, briefly skim through the documentation, as this will give you a better idea of who this service is meant for and how you can make the best of it. To begin, try automatically detecting starting points (just press submit). This might give you a clue where there are channels in your structure, and then you can select those closest to the site of your interest (i.e. active site, cofactors, etc.) or try to select endpoints of the pore by clicking on the molecular surface. Alternatively, you can load any channels deposited in the ChannelsDB 2.0.

Here, we provide several tips on how some of the implemented algorithms' limitations could also be overcome.

• The first limitation is that the tunnel is shown only as balls of maximum size along the centerline. This limitation can be overcome when starting points are put along the channel to find whether there are bulges along the central line. However, the centerline provides a good measure of where the channels are localized in the structure.

• The second limitation is the use of atom-centered Voronoi mesh. It isn't easy to overcome, and the real improvement is debatable. The atom-centered Voronoi mesh can be replaced, e.g., by a power diagram, which will improve precision. However, such improvement is small compared to the uncertainties associated with the chosen structures (e.g. X-ray structures with finite resolution, generally higher than 0.8 Å).

However, we are certain that a user can face many problems trying to analyze his/her specific molecule. We are ready to help users and provide feedback via email: [email protected] to resolve specific problems.

Several reasons can cause this. Always remember that we are happy to help you with any issues you might encounter, so don’t be shy and ask for help! Here is a list of the most common issues:

1. The setup of ProbeRadius, InteriorThreshold, or filtering criteria was wrong.

Setting up the two key parameters can be, at times, critical for the proper identification of channels. These parameters can be considered a lower bound (InteriorThreshold) and an upper bound (ProbeRadius) threshold for a channel radius. Therefore, if the expected channel is supposed to be wide in its radius, the ProbeRadius parameter must be increased, preferably above the expected channel radius. Likewise, if the channel is expected to pass through a constringent region with a local narrowing, the InteriorThreshold must be set below the expected minimal channel radius.

Finally, all the channels are the subject of the filtering step. Try adjusting the values of MaxTunnelSimilarity, BottleneckRadius or BottleneckLength. It is always good practice to set BottleneckRadius a little lower than InteriorThreshold and allow BottleneckLength to cover for small local narrowings beyond this threshold.

Example:

2. The location of the active site (channel starting point) is wrong.

Even if the two major parameters are set correctly, the deemed channels still cannot be seen in certain cases. The MOLE algorithm is designed to identify channels leading to the buried sites inside the protein structure. Therefore, if your active site is located in a shallow pocket or on the protein surface, the channel analysis will not help here.

Alternatively, if your active site is buried within a protein body, you can enlarge the space to search for potential channel starts by increasing the OriginRadius parameter.

Example:

3. Substrate is blocking the channel.

MOLE, in its default settings, considers only protein/DNA/RNA atoms, effectively discarding any het groups and solvent molecules before the calculation. However, even this can be modified to keep, for instance, cofactors in the structure before the calculation. The algorithm heavily relies on the correct annotation in the input structure. That is the annotation of the _atom_site.group_PDB field, which is set to either ATOM or HETATM in the mmcif/PDB language.

Example:

In case you are required to switch off a certain part of the structure (residues or even certain atom groups) before the calculation. You can select those in the active atoms/residues section. We strongly encourage you to try PatternQuery syntax, the most flexible option. For example, to discard all the alanine residues, you type in: Residues('ALA'). To discard the ammonium group of lysine residues capable of carrying protons, simply add: AtomNames('NZ').Inside(Residues('LYS')). If you want to combine both, just join them to: Or(Residues('ALA'), AtomNames('NZ').Inside(Residues('LYS'))).

4. There is no channel at all.

The final option, if none of the above helped. There is always a possibility that there are no reasonably sized channels in the protein conformation you provided. Drop us an email: [email protected] so we can look at whether this is the case!

Give it a go in the pore mode. This is the most comfortable option for most transmembrane molecular pores, as it requires almost no setup. After the calculation, check the position of the membrane. If the position is not of biological relevance, the pore will not be found, and it will likely be wrong. If the pore module fails, please let us know. There is always room for improvement.

In most of the transmembrane channel structures, the putative centerline can be seen by the naked eye. In this case, switch back to the channels mode, display the cavity from the upper right panel, and select pore endpoints using the Ctrl + left click option. Lastly, identify them as channel endpoints in the menu. Do not forget to change the parameters of ProbeRadius and InteriorThreshold, as those differ in Pore and Channels mode.

If the channel is large enough to be visible to the naked eye, it can be larger, usually shown as a part of the surface. Consequently, it is necessary to enlarge the Probe radius to cover such a structure.

When the channel is too narrow in a certain part, it sometimes helps to lower the InteriorThreshold parameter.

MOLEonline enables analysis of some basic physicochemical properties - hydropathy, hydrophobicity, polarity, lipophilicity, solubility and mutability, lipophilicity (logP and logD), water solubility (logS-like scale of channel-lining residues). This is a step forward in channel analysis. Still, we are certain that it will take some time to optimize the analysis of these features and to understand their biological relevance fully. Considering all the pros and cons, we decided to keep this functionality in the current version to enable “experiments” with this feature for a broad scientific community.

Every amino acid can be mutated; however, some amino acids can be mutated more safely concerning a protein's structure/stability/folding. The relative mutability index reflects this feature, e.g. PRO and TRP residues are typically structurally important, and their mutations often lead to unstable proteins or proteins with alternated structures. The relative mutability index which we employ is based on the analysis of the protein sequences (similar to well-known BLOSUM and PAM matrices used in sequence alignments), and it was taken from (Jones1992).