Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction Academic Article uri icon

abstract

  • The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10-100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of the dependence of crystal quality on crystal size.

authors

  • Abdallah, Bahige G
  • Zatsepin, Nadia A
  • Roy-Chowdhury, Shatabdi
  • Coe, Jesse
  • Conrad, Chelsie E
  • Dörner, Katerina
  • Sierra, Raymond G
  • Stevenson, Hilary P
  • Camacho-Alanis, Fernanda
  • Grant, Thomas D
  • Nelson, Garrett
  • James, Daniel
  • Calero, Guillermo
  • Wachter, Rebekka M
  • Spence, John CH
  • Weierstall, Uwe
  • Fromme, Petra
  • Ros, Alexandra

publication date

  • 2015