Fig. 5: Structural analysis and comparison of urea transport in zf-UT and hUTs.

a, b Overall view of the urea density in zf-UT trimer. Two portions of obvious and continuous cryo-EM densities corresponding to urea in zf-UT are located at both the intracellular and extracellular sides of the channel (a), which facilitated the unambiguous assignment of urea molecules (b). c Comparison of the transportation channel of zf-UT (blue) and hUT-A2 (green). According to the EM density distribution, the urea transportation channel of zf-UT contained a vertical tubular cavity composed by 3a, 3b, 5a, 5b, Pa, and Pb helices, and then extends into the intracellular section composed by intracellular ends of helices 3a–5a and ICL2b. d Free energy surface (FES) of urea across the zf-UT channel, revealing six low-energy states-So’, So, Sm, Si, Si’, and Si”-associated with extended urea residence times across six distinct regions within the channel. e Simplified one-dimensional energy landscape illustrating the So’, So, Sm, Si, Si’, and Si” states of urea within the zf-UT channel. f The location of the urea molecules are modeled into six regions shown as So’, So, Sm, Si, Si’, and Si” in the zf-UT channel surrounding with presentation of key interacting residues. The urea transport channel of zf-UT is depicted by white dotted line with the inner urea molecules displayed as stick-balls. The conserved Q^Pb–T^5b–T^5a–Q^Pa motif is shown as green dots. g, h The interactions between urea and the key residues in the zf-UT urea transport channel (shown as blue dots) in the extracellular side (g) and the intracellular side (h) of the channel. The H-bonds are shown as red dashed line. i Barcode comparisons of the residues involving in the urea channel of zf-UT between different UTs. The residues predicted to involve with common interactions with urea molecules are displayed as black circles, including residues of the Q^Pb–T^5b–T^5a–Q^Pa motif filled with green, while the residues predicted to form distinct interactions are shown as black circles filled with gray. The letters inside the circles represent different amino acids present in the corresponding positions.