The mouse serotonin 5-HT3A receptor is a homo-pentameric ligand-gated ion channel (pLGIC) mediating fast excitatory neurotransmission in the central nervous system. The molecular mechanism of ion permeation of 5-HT3A receptors triggered by the neurotransmitter serotonin is not yet fully understood. The recent X-ray structure of the mouse serotonin 5-HT3A receptor in complex with a stabilizing nanobody revealed for the first time the entire structure of a mammalian pLGIC in detergent. Structural information of the receptor in a lipid bilayer however is still limited primarily due to the lack of 2D crystals of the receptor in a lipid bilayer. Here we present our results on the formation and improvement of diffracting 2D crystals of the mouse 5-HT3A by limited proteolysis and addition of conformational nanobodies. (C) 2016 Elsevier Ltd. All rights reserved.