4.0 Article Proceedings Paper

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

Publisher

INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S2053230X15009966

Keywords

batch crystallization; GPCR; serial crystallography; FEL; dynamics

Funding

  1. SNF [R'EQUIP145023, 31003A_141235, 310030_153145, 31003A_146520]
  2. Swiss National Foundation through NCCR MUST
  3. PIER Helmholtz Graduate School
  4. Helmholtz Association
  5. NSF STC award
  6. NSF [1231306]
  7. NIH [GM097463-04]
  8. [FP7-PEOPLE-2011-ITN 317079 NanoMem]
  9. Swiss National Science Foundation (SNF) [31003A_146520] Funding Source: Swiss National Science Foundation (SNF)

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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.

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