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Title: 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 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 in this study, 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.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Paul Scherrer Inst., Villigen (Switzerland). Lab. for Biomolecular Research
Publication Date:
Type:
Accepted Manuscript
Journal Name:
Acta Crystallographica. Section F, Structural Biology Communications
Additional Journal Information:
Journal Volume: 71; Journal Issue: 7; Journal ID: ISSN 2053-230X
Publisher:
International Union of Crystallography
Research Org:
Paul Scherrer Inst., Villigen (Switzerland). Lab. for Biomolecular Research
Sponsoring Org:
USDOE; National Institutes of Health (NIH); National Science Foundation (NSF)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; batch crystallization; GPCR; serial crystallography; FEL; dynamics
OSTI Identifier:
1345038