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Updated: Apr 7, 2026
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Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Published on: September 20, 2016
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Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser
Wenting Wu1, Przemyslaw Nogly1, Jan Rheinberger1
1Laboratory for Biomolecular Research, Paul Scherrer Institute, OFLC/103, 5232 Villigen-PSI, Switzerland.
Summary
Researchers developed a method for producing high-density microcrystals of rhodopsin, a key protein for night vision. This advancement enables ultrafast dynamics studies using X-ray free-electron lasers (XFELs).
Area of Science:
- Structural biology
- Biophysics
- Crystallography
Background:
- Rhodopsin, a G protein-coupled receptor, forms the visual pigment with retinal, crucial for night vision.
- Ultrafast dynamics studies of rhodopsin's non-reversible activation require time-resolved serial femtosecond crystallography (TR-SFX).
- TR-SFX necessitates microcrystals delivered into X-ray pulses after precise photoactivation delays.
Purpose of the Study:
- To develop a method for milliliter-scale production of high-density rhodopsin microcrystals.
- To enable serial femtosecond crystallography (SFX) studies of rhodopsin dynamics.
Main Methods:
- Adapted known vapor-diffusion crystallization protocols.
- Screened low-salt conditions suitable for serial crystallography.
- Optimized batch crystallization and assessed crystal quality using SHG imaging and X-ray powder diffraction.
- Produced milliliter batches of rhodopsin microcrystal suspension for SFX tests.
Main Results:
- Successfully developed a four-step protocol for milliliter batch production of rhodopsin microcrystals.
- The produced microcrystals exhibited suitable size and quality for serial crystallography.
- Rhodopsin microcrystals diffracted X-rays at an X-ray free-electron laser (XFEL) facility using a liquid-jet setup.
Conclusions:
- A scalable method for producing rhodopsin microcrystals for time-resolved studies was established.
- This work facilitates advanced investigations into the ultrafast dynamics of rhodopsin activation.
- The developed protocol supports the application of XFELs for studying membrane protein dynamics.
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