{"id":5831,"date":"2025-12-04T11:22:04","date_gmt":"2025-12-04T11:22:04","guid":{"rendered":"https:\/\/www.sygnaturediscovery.com\/?post_type=case-study&p=5831"},"modified":"2026-02-05T16:55:59","modified_gmt":"2026-02-05T16:55:59","slug":"determining-the-cryo-em-structure-of-the-5-ht2a-receptor-with-serotonin-bound","status":"publish","type":"case-study","link":"https:\/\/www.sygnaturediscovery.com\/fr\/case-study\/determining-the-cryo-em-structure-of-the-5-ht2a-receptor-with-serotonin-bound\/","title":{"rendered":"Determining the Cryo-EM Structure of the 5-HT2A receptor with Serotonin Bound"},"content":{"rendered":"\n

In this case study,\u00a0Rachel Johnson<\/a>\u00a0describes how we determined the active state structure of the 5-HT2A<\/sub>\u00a0receptor with the natural ligand serotonin bound using cryo-EM.<\/h2>\n\n\n\n

Introduction<\/h3>\n\n\n\n

GPCRs are a large and diverse group of receptors in the body. They are also important therapeutic targets as over a third of all FDA approved drugs act upon these receptors [1]. Being able to probe how ligands interact with the target protein by elucidating the structural details of how the ligand binds to the protein and\/or revealing any changes to the conformation of the protein can significantly impact drug discovery programs. Cryo-EM has become a powerful tool in the structure determination of GPCRs [2]. The resolutions attained have allowed small molecule ligands to be visualised within the maps and have also revealed different receptor conformations upon ligand binding and enabled receptor dynamics to be studied [3, 4].<\/p>\n\n\n\n

The 5-hydroxytryptamine receptor isoform 2A (5-HT2A<\/sub>R) is one of a family of GPCRs activated by serotonin (5-hydroxytryptamine) and is essential for learning and cognition [5]. Pathophysiology associated with 5-HT2A<\/sub>R has been linked to psychiatric disorders and conditions including depression, schizophrenia, and drug addiction. In addition to serotonin, 5-HT2A<\/sub>R is also the target for agonists that possess cognition-enhancing and hallucinogenic properties, such as LSD and psilocin. On the other hand, 5-HT2A<\/sub>R antagonists have antipsychotic and antidepressant properties. Thus, 5-HT2A<\/sub>R has seen recent focus as a possible target in the treatment of a range of neuropsychiatric conditions [6]. Here, we aimed to determine the active state structure of 5-HT2A<\/sub>R with the natural ligand serotonin bound using cryo-EM.<\/p>\n\n\n\n

Sample preparation<\/h3>\n\n\n\n

Initially, we co-expressed the receptor and G proteins in Sf21 insect cells using the baculovirus expression system where the components of the complex (5-HT2A<\/sub>R, G\u03b1q, G\u03b21 & Gg2) were added at a 1:1:1:1 ratio. Following an incubation with serotonin, the complex was purified from membranes via FLAG affinity and size exclusion chromatography (SEC). Sample quality is crucial for cryo-EM, so final QC checks were performed. SDS-PAGE and MS-MS peptide mapping was used to ensure all complex components were present. An analytical SEC was also performed to check that the protein could withstand a freeze-thaw cycle. This is important because if complexes are unstable during freeze-thaw, they must be applied to a cryo-EM grid immediately after purification for optimal structure determination. QC checks for5-HT2A<\/sub>R confirmed all components of the complex were present in the final sample and that the complex survived a freeze-thaw cycle. Therefore, the sample was taken forward for imaging, and frozen sample could be used.<\/p>\n\n\n\n

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Figure 1:<\/strong>\u00a0Protein purification and quality control of the 5-HT2A<\/sub>R sample for cryo-EM. Fractions from the preparative SEC trace peak were pooled. The freeze-thaw and SDS-PAGE QC checks showed that the all components of the complex were present and that it could withstand a freeze-thaw cycle.<\/em><\/figcaption><\/figure>\n\n\n\n

Cryo-EM structure determination<\/h3>\n\n\n\n

Cryo-EM grids of the 5-HT2A<\/sub>R complex were prepared using a ThermoFisher Vitrobot Mark IV. 3 \u00b5L of complex was applied to a Quantifoil R1.2\/1.3 Cu 400 mesh grid, excess sample was blotted off and the grid was plunged into liquid ethane rapidly freezing the sample in a thin layer of vitrified ice to preserve its native state. Prepared grids were screened for good particle distribution in thin ice. Suitable regions of the grid were identified and a data set consisting of ~11,000 movies was collected on the Titan Krios microscope fitted with a K3 detector at the Midlands Regional Cryo-EM Facility. After motion correction and CTF refinement, particles were picked using Topaz [7] and classified in 2D using Relion5 [8]. The particles which contributed to good 2D averages that displayed clear secondary structure were then used for 3D refinement and subjected to particle polishing which resulted in a 3.7\u00c5 map being obtained. Atomic coordinates were then fitted into the polished map and subjected to real space refinement.<\/p>\n\n\n\n

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