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

Acta Crystallogr F Struct Biol Commun. 2015 Jul;71(Pt 7):856-60. doi: 10.1107/S2053230X15009966. Epub 2015 Jun 27.

Abstract

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.

Keywords: FEL; GPCR; batch crystallization; dynamics; serial crystallography.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Crystallization
  • Lasers / statistics & numerical data*
  • Rhodopsin / chemistry*
  • X-Ray Diffraction / methods*

Substances

  • Rhodopsin