Hi all,

We’re currently exploring whether a particular molecule enters cells via passive diffusion across the lipid bilayer or through endocytosis. To probe this, we’re using MD simulations to assess its membrane permeability.

In our unbiased simulations, we observe repeated binding and unbinding events between the molecule and the membrane surface, but no leaflet-crossing events — the molecule never traverses from the top leaflet to the bottom and exits the membrane.

We’re wondering:

• Are bilayer-crossing events typically rare in unbiased simulations due to high energetic barriers?
• Is there a recommended unbiased strategy to observe these events, or would enhanced sampling (e.g., umbrella sampling) be more appropriate?
• If we go the umbrella sampling route, are there reference systems or standards that are commonly used for benchmarking small molecule permeability?

We haven’t been able to find published examples where unbiased MD alone was sufficient to estimate passive permeation of small molecules, so any pointers (papers, parameters, collective variables, etc.) would be greatly appreciated.

Thanks in advance!