skip to main content


Title: Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

We report that 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 canmore » 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. Ultimately, this method will also permit an analysis of the dependence of crystal quality on crystal size.« less
 [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [3] ;  [4] ;  [5] ;  [6] ;  [7] ;  [2] ;  [2] ;  [5] ;  [6] ;  [2] ;  [2] ;  [1] ;  [1]
  1. Arizona State Univ., Tempe, AZ (United States). Dept. of Chemistry and Biochemistry; Arizona State Univ., Tempe, AZ (United States). Biodesign Inst.
  2. Arizona State Univ., Tempe, AZ (United States). Biodesign Inst.; Arizona State Univ., Tempe, AZ (United States). Dept. of Physics
  3. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany)
  4. SLAC National Accelerator Lab., Menlo Park, CA (United States). Photon Ultrafast Laser Science and Engineering Inst. (PULSE)
  5. Univ. of Pittsburgh School of Medicine, Pittsburgh, PA (United States). Dept. of Structural Biology
  6. Arizona State Univ., Tempe, AZ (United States). Dept. of Chemistry and Biochemistry
  7. Hauptman-Woodward Medical Research Inst. (HWI), Buffalo, NY (United States)
Publication Date:
Grant/Contract Number:
R01GM095583; R01GM112686; R01GM097463; AC02-76SF00515; 1231306
Accepted Manuscript
Journal Name:
Structural Dynamics
Additional Journal Information:
Journal Volume: 2; Journal Issue: 4; Journal ID: ISSN 2329-7778
American Crystallographic Association/AIP
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; 36 MATERIALS SCIENCE; Proteins; Crystal Structure; X-ray diffraction; Suspensions; Nanocrystals
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1421055