Extended Data Table 1:

Sequence of mouse 5-HT_3AR used in the cryo-EM study and the data on cryo-EM and refinement. a, Full length mouse 5-HT_3AR sequence used in the cryo-EM study. Regions in the sequence highlighted in green, blue, gray, and yellow represent strep-tag, linker, TEV cleavage site, and 1D4-tag, respectively. Secondary structural elements as seen in State 1 are plotted above the sequence. Loops in gray color are not seen in the final refined structure. All the important loops, sheets, and helices are labeled. Glycosylation sites are marked as blue arrows. Key residues within the serotonin-binding sites are highlighted in brown color. Cysteines present in the cys-loop are shown as cyan color. Pore-facing residues in M2 are shown in green color. Arg416 in the ICD is shown in red. b, Cryo-EM data collection, refinement and validation statistics.

a
b		State 1 (EMDB-7882) (PDB 6DG7)	State 2 (EMDB-7883) (PDB 6DG8)
	Data collection and processing
	Magnification	130,000x
	Voltage (kV)	300
	Electron exposure (e-/Å2)	40
	Defocus range (µm)	−1.0 to −2.5
	Pixel size (Å)	0.532
	Symmetry imposed	C5
	Initial particle images (no.)	749,970
	Final particle images (no.)	103,698	18,839
	Map resolution (Å)	3.32	3.89
	FSC threshold	0.143	0.143
	Refinement
	Initial model used (PDB code)	6BE1	6BE1
	Model resolution (Å)	4.31	4.31
	FSC threshold	0.143	0.143
	Map sharpening B factor (Å2)	−50	−50
	Model composition
	Non-hydrogen atoms	16,720	16,715
	Protein residues	16,175	16,175
	Ligands	545	540
	B factors (Å2)
	Protein	154.41	244.56
	Ligand	133.45	169.20
	R.m.s. deviations
	Bond lengths (Å)	0.004	0.004
	Bond angles (°)	1.032	1.031
	Validation
	MolProbity score	1.53 (94th Percentile)	1.56 (94th Percentile)
	Clashscore	3.40 (97th Percentile)	4.89 (94th Percentile)
	Poor rotamers (%)	1.27	0.55
	Ramachandran plot
	Favored (%)	95.61	95.61
	Allowed (%)	4.39	4.39
	Disallowed (%)	0.00	0.00