The manuscript by Pedersen et al. with the title “Cryo-EM structure of the dopamine transporter with a novel atypical non-competitive inhibitor bound to the orthosteric site” describes the structure determination of AC-4-248 bound to a termostabilized dDAT, experimental verification of key pharmacological parameters and of a few crucial mutants. The study concludes with docking predictions of binding pose, especially of the trimeric complexes of dDAT/AC-4-248/cocaine or dopamine.

The manuscript is well written and clear to follow.

My main concert is the apparent non-competitive behavior:
The non-competitive behavior of AC-4-248 and DA seems difficult to reconcile with the outward open structure. This study shows that AC-4-248 stabilizes dDAT in the outward open conformation and in hDAT, the Km of dopamine (DA) is 1.5 μM, while the IC50 of AC-4-248 is 45.5 μM, while in dDAT, the IC50 of AC-4-248 is 90 μM. Given the observed outward open conformation of DAT and similar values of Km and IC50, one would expect a competitive behavior similar to cocaine and DA. Given the importance given to the competition through the docking study, a potential origin of the non-competitive behavior needs to be discussed.

Minor concerns

-Figure 2c: Please consider label the helices in the two zoom in images.
-Figure 3a: Please label the helices

-Figure 3d: The legend for figure 3d says: “The central binding pocket comprising subsites A, B, and
C are shown from the top and side in the outward open, AC-4-248-bound dDAT structure.” I can see three images, not two. Most likely one top and two side view. Please indicate correctly what the three images show. Also, please label the helices. In the current picture it is impossible to correctly link the binding pockets A,B,C and the bound compounds to their respective binding pose in dDAT. Maybe showing a few side chains would further help.

-Supplementary Figure S5a: The degree of conservation between dDAT and hDAT is very difficult to grasp from the figure. The property describe by the color code is not very clear. Intuitively, I would expect: yes/no for being identical, not a scale. The legend say conservation score, but I guess it is more a similarity score for differing residues. Needs some explanation. The authors might want to consider showing the sequence alignment of dDAT and hDAT to help the reader to appreciate residues identity and differences.
Some residues are highlighted by black squares, but it remains unclear, what is highlighted.

• Discussion
  The authors write:“An alternative approach could involve utilizing low-affinity inhibitors of DAT, potentially reducing the likelihood of abusive behavior and alleviating withdrawal symptoms”
  The author might want to consider extending the first paragraph by describing, why a low-affinity inhibitor could potentially reduce the abusive behavior and alleviating withdrawal symptoms.

• Methods
  --Thermal shift assay. Please describe which sample preparation condition were used.
  --There seems to be an unintended number in the first line of describing of XP docking. Please check.

• wwPDB validation report
  The headline and the watermark of each page says: “Not For Manuscript Review”. Though very useful information, it is unclear, why was this document added to the SI

The authors have successfully addressed all my concerns in the revised version of their manuscript.

The author have successfully addressed all my concerns in the previous revised version of their manuscript.