Where we think our MD may really inform the pH discus-415 sion is in noting that many water molecules can readily transit 416 through the carboxysome shell (Table , SI Fig. ). Hydrogen 417 bond arrangements that could conduct a proton across the 418 shell through a Grothuss-type mechanism postulated previ-419 ously are trivial to identify from our trajectories. Thus 420 any pH gradient cannot be manufactured by active transport 421 processes, as the gradient would be run down by these Grothuss 422 proton movements, and lead to proton movements observed 423 in prior experiments and systems modeling predictions 424 . Instead, based on the electrostatic surface analysis of 425 the synthetic shell reveals a more negatively charged exterior 426 relative to the interior , and is a lever evolution can turn to 427 optimize the interior pH of a carboxysome by modifying the 428 Nernst potential relative to the cytosol, as well as the metabo-429 lite permeability. Mutation is the clearest path forward to 430 modify any potential pH gradient, as the carboxysome is too 431 small for the number of free protons to be a useful indicator 432 of pH. An approximately spherical 120 nm diameter solvated 433 carboxysome will hold only 30.3 million water molecules * , 434 thus even an empty shell will have fewer than 1 H3O + in its 435 interior at pH 7. Instead, the pH environment within the 436 carboxysome is fixed by the protonation equilibria of the shell 437 and its cargo, which in our fixed charge simulation models 438 take the predominant protonation state at pH 7.