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

Structural Dynamics
Bahige G AbdallahAlexandra Ros

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 a...Continue Reading

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Citations

Dec 13, 2016·Analytical Chemistry·Paul V JonesAlexandra Ros
Aug 17, 2018·Electrophoresis·Blanca H Lapizco-Encinas
Mar 2, 2019·Acta Crystallographica. Section D, Structural Biology·Marie Luise Grünbein, Gabriela Nass Kovacs
Jan 31, 2019·Annual Review of Analytical Chemistry·Mukul SonkerAlexandra Ros
Oct 23, 2019·Journal of Applied Crystallography·Austin EchelmeierAlexandra Ros
May 4, 2016·Acta Crystallographica. Section D, Structural Biology·Hilary P StevensonGuillermo Calero
Dec 5, 2019·Journal of Applied Crystallography·John H BealeRobin L Owen
Oct 14, 2020·Current Opinion in Structural Biology·Allen M Orville
Nov 3, 2020·Beilstein Journal of Nanotechnology·Daniel F QuevedoBlanca H Lapizco-Encinas
Nov 30, 2018·Analytical Chemistry·Daihyun KimAlexandra Ros

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Methods Mentioned

BETA
X-ray
dynamic
electron microscopy
fluorescence imaging
dynamic light scattering
fluorescence microscopy
light scattering

Software Mentioned

SONICC
LabSmith μScope
sim
AutoCAD
Nanoparticle Tracking Analysis ( NTA )
RIP
pattern
Manager
HI
CrystFEL

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