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Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser

Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the n...

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Detalles Bibliográficos
Autores principales: Wu, Wenting, Nogly, Przemyslaw, Rheinberger, Jan, Kick, Leonhard M., Gati, Cornelius, Nelson, Garrett, Deupi, Xavier, Standfuss, Jörg, Schertler, Gebhard, Panneels, Valérie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498706/
https://www.ncbi.nlm.nih.gov/pubmed/26144230
http://dx.doi.org/10.1107/S2053230X15009966
Descripción
Sumario:Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.