| Summary: | 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.
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