The mechanosensitive channel YbiO has a conductance equivalent to the largest gated pore

Bacterial mechanosensitive channels are divided into large (MscL) and small (MscS-like) conductance families. The function of MscS and MscL is to protect cells against osmotic shock by acting as pressure safety valves. Within the MscS-like family, E. coli encodes much larger channels, such as YbiO, MscK, and MscM, but their physiological role remains unclear. Compared to MscL their conductances are reported as 3–10 times lower. We show that YbiO can achieve a conductance of ∼3 nS, and an equivalent pore opening of > 25 Å in diameter, equaling the known largest gated pore, MscL. We determine a cryoelectron microscopy (cryo-EM) structure of YbiO in a sub-open conformation, demonstrating the existence of multiple substates. One substate is consistent with the pore opening extent of our structure and the other matches states previously thought to resemble full openings. Our findings demonstrate surprising capabilities, hinting at new physiological roles for YbiO and potentially other MscS-like channels.