Home › Pharmacology › Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Pharmacology JoVE (Open Access) Citable · DOI

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

DOI: 10.3791/54463-v
What you'll learn
  • ✓ Prepare membrane protein microcrystals in lipidic cubic phase
  • ✓ Deliver microcrystals for serial femtosecond crystallography experiments
  • ✓ Reduce sample consumption versus conventional liquid injection methods
  • ✓ Characterize microcrystal quality before synchrotron/XFEL delivery
Protocol

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Difficulty
advanced
Total time
~3–5 days (crystallization setup to final delivery and data collection)

Steps

1
Set up crystallization in syringes and detect crystals

Load protein solutions into syringes and initiate crystallization trials. Monitor syringe contents to identify crystal formation under experimental conditions.

▶ 00:54
2
Consolidate samples and titrate with monoacylglycerol

Harvest and consolidate microcrystal samples, then titrate with 7.9 MAG to form the lipidic cubic phase matrix for delivery.

▶ 04:06
3
Characterize microcrystals for quality assessment

Evaluate microcrystal morphology, size distribution, and phase homogeneity to confirm suitability for serial crystallography experiments.

▶ 06:32
4
Validate sample preparation and collect diffraction data

Deliver prepared microcrystals to X-ray free-electron laser or synchrotron beamline and record representative protein structures from serial crystallography measurements.

▶ 07:44
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