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

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

doi:10.1063/1.4928688

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.

Highlights

X-ray free-electron laser (XFEL) technology has become popular over recent years in the field of protein crystallography.1–3 This technology was proposed to facilitate structural studies of difficult-to-crystallize proteins4–6 that failed to produce crystals large enough for traditional synchrotron-based crystallography where the crystal is exposed to the X-ray beam for durations longer than the onset of detrimental radiation damage
We further describe the second-generation microfluidic sorting device, provide a detailed sorted sample characterization using several methods, including dynamic light scattering (DLS), NanoSight particle tracking, and electron microscopy (EM), and examine diffraction patterns obtained from sorted protein nanocrystals
Our XFEL crystal size optimization is based on sorting a bulk photosystem I (PSI) crystal suspension by size to isolate submicron fractions with reduced size dispersity using microfluidics

Summary

INTRODUCTION

X-ray free-electron laser (XFEL) technology has become popular over recent years in the field of protein crystallography. This technology was proposed to facilitate structural studies of difficult-to-crystallize proteins that failed to produce crystals large enough for traditional synchrotron-based crystallography where the crystal is exposed to the X-ray beam for durations longer than the onset of detrimental radiation damage. Because SFX is a serial method where a new crystal is brought into the X-ray interaction region for each X-ray pulse, patterns from many different crystals must be indexed and their structure factors merged to reconstruct the electron density map of the molecules in the unit cell This analysis effectively performs a Monte Carlo integration over a (typically) heterogeneous distribution of crystal sizes, shapes, and orientations, as well as the stochastically varying XFEL pulse intensity and spectrum.. Sorted PSI nano- and microcrystal fractions were prepared for SFX experiments at LCLS by first concentrating them 3–4 fold by centrifugation (

RESULTS AND DISCUSSION

CONCLUSIONS

Methods