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Nat Commun ; 12(1): 1074, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594077


Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT3A serotonin receptor (5HT3R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT3R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, 'coupled' state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT3R by the membrane environment, and a model for asymmetric activation of the receptor.

Bicamadas Lipídicas/metabolismo , Multimerização Proteica , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Linhagem Celular , Microscopia Crioeletrônica , Lipídeos/química , Camundongos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores 5-HT3 de Serotonina/ultraestrutura , Serotonina/farmacologia
Nat Commun ; 11(1): 3709, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709843


Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.

Canais de Cálcio/química , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Tomografia com Microscopia Eletrônica/métodos , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Razão Sinal-Ruído , Imagem Individual de Molécula , Fluxo de Trabalho