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Droplet streams for serial crystallography of proteins

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Abstract

Serial diffraction of proteins requires an injection method to deliver analyte molecules—preferably uncharged, fully hydrated, spatially oriented, and with high flux—into a focused probe beam of electrons or X-rays that is only a few tens of microns in diameter. This work examines conventional Rayleigh sources and electrospray-assisted Rayleigh sources as to their suitability for this task. A comparison is made and conclusions drawn on the basis of time-resolved optical images of the droplet streams produced by these sources. Straight-line periodic streams of monodisperse droplets were generated with both sources, achieving droplet diameters of 4 and 1 micrometer, respectively, for the conventional and electrospray-assisted versions. Shrinkage of droplets by evaporation is discussed and quantified. It is shown experimentally that proteins pass undamaged through a conventional Rayleigh droplet source.

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Acknowledgments

This work was supported by NSF award IDBR 0555845 and ARO award DAAD190010500.

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Authors and Affiliations

  1. Department of Physics, Arizona State University, Tempe, AZ, 85287, USA

    U. Weierstall, R. B. Doak, J. C. H. Spence, D. Starodub, P. Kennedy, J. Warner & G. G. Hembree

  2. Advanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA

    D. Shapiro

  3. Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287, USA

    P. Fromme

  4. Lawrence Livermore National Laboratory, 7000 East Ave., Livermore, CA, 94550, USA

    H. N. Chapman

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  1. U. Weierstall
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Appendix: Coherence and alignment

Appendix: Coherence and alignment

Ideally, the dilution of the analyte solution is set to deliver no more than a single protein per microdroplet. This is not a major constraint, however. Provided the coherence patch of the probe beam encompasses at most one molecule, the diffraction pattern is a simple sum of intensities from the individual molecules and the pattern from N molecules is identical to that of a single molecule, just scaled up by a factor of N. If the coherence patch encompasses two or more molecules, then intra-particle interference does result and diffraction amplitudes add rather than intensities. However, this interference either averages to zero over the exposure (multiple proteins at random spacing within one or more droplets) or can be separated out by virtue of a disparate length scale (exactly-spaced proteins in separate droplets of a perfectly periodic droplet stream). Coherence can often be traded for flux in an X-ray source, and so the coherence patch for a given experiment should be set to just larger than the protein size in order to work at the highest possible flux.

At the near-IR laser frequency to be used for molecular alignment, biomolecules can be treated as approximately homogeneous bodies with dielectric constant of 2–2.3 (Arakawa et al. 1997, 2001) This being the case, the induced dipole moment of a protein is mostly due to the shape anisotropy of the molecule. At low frequency, polarization is due to the re-orientation of individual polar groups within the protein. Given its homogeneity and low dielectric constant, the interior contributes little. In contrast, a large polarization arises on the highly inhomogeneous surface due to the side-chain re-orientation (Simonson 2003). In a DC E-field, permanent electric dipole moments (which cancel out in an AC laser field) can also produce large alignment forces. Thus strong electrostatic (Koch et al. 1988) and magnetic fields (Bras et al. 1998) might also be used alone or in combination with CW laser fields to promote alignment. Flow alignment due to hydrodynamic shear is also possible, as used in studies of electric birefringence, and this may well be expected in the Poiseuille-like flow of droplet stream nozzles.

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Weierstall, U., Doak, R.B., Spence, J.C.H. et al. Droplet streams for serial crystallography of proteins. Exp Fluids 44, 675–689 (2008). https://doi.org/10.1007/s00348-007-0426-8

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