Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers


X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

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Figure 1: Experimental setup.
Figure 2: Photo-initiated XES of PS II.
Figure 3: XES of gas-activated RNR.
Figure 4: XRD of various enzymes.

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We thank T. Rendahl from LCLS for help with controls and B. Martins, S. Myers, M. Cowan, G. Shea-McCarthy and C. Whalen (Brookhaven National Laboratory) for help with engineering and controls, R. Ellson, J. Olechno, R. Stearns and B. Hadimioglu (LABCYTE) for sharing their experience with the acoustic transducers and related issues, C. Saracini (LBNL) for his help with the preliminary testing of the DOT system, P. Glatzel (ESRF) for discussion of the Fe Kα emission and F. Houle (LBNL) for sharing her reaction–diffusion simulation software Kinetiscope and for useful discussions. This work was supported by the Director, Office of Science, Office of Basic Energy Sciences (OBES), Division of Chemical Sciences, Geosciences and Biosciences (CSGB) of the US Department of Energy (DOE) under contract DE-AC02-05CH11231 (J.Y. and V.K.Y.) for X-ray methodology and instrumentation, by the US National Institutes of Health (NIH) grants GM110501 (J.Y.) for instrumentation development for XFEL experiments, GM102520 (N.K.S.) and GM117126 (N.K.S.) for development of computational protocols for XFEL data and GM055302 (V.K.Y.) for PS II biochemistry, structure and mechanism, a Ruth L. Kirschstein National Research Service Award (5 F32 GM116423-02; F.D.F.), the Human Frontiers Science Project award no. RGP0063/2013 310 (J.Y., U.B. and A.Z.), and the Science and Technology Center program of the US National Science Foundation (NSF) through BioXFEL under agreement no. 1231306 (J.A.C., M.D.M. and G.N.P.). R.D.V. is supported by NSF grant MCB-1329956. J.A.C. was supported by a training fellowship from the Gulf Coast Consortia on the Houston Area Molecular Biophysics Program (NIHGMS grant no. T32GM008280). C.J.P. was supported by NIH NRSA grant GM113389-01. Portions of this work were supported by Brookhaven National Laboratory (BNL)-US DOE, Laboratory Directed Research and Development grant 11-008 (C.G.R., M. Allaire., A.M.O.), NIH–NCRR grant 2-P41-RR012408 (A.M.O. and M. Allaire), NIH–National Institute of General Medical Sciences (NIGMS) grants 8P41GM103473-16 (A.M.O.) and Y1GM008003 (M. Allaire) and US DOE, Office of Biological and Environmental Research (OBER) grant FWP BO-70 (A.M.O. and B.A.). A.M.O., P.A. and P.T.D. were supported in part by Diamond Light Source, and A.M.O. acknowledges support from a Strategic Award from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council (grant 102593). P.B. was supported by a Wellcome Trust DPhil studentship. M.H. received support from the Knut and Alice Wallenberg Foundation, the Swedish Cancer Society, the Wenner–Gren foundations and the Swedish Research Council (grants 2013-541 and 2013-5884). C.A.S. acknowledges support from the US DOE, Office of Science, Division of CSGB. A.Z. acknowledges support from the DFG-Cluster of Excellence 'UniCat', coordinated by the Technische Universität Berlin, and Sfb1078 (Humboldt Universität Berlin), TP A5. J.M. acknowledges support from the Solar Fuels Strong Research Environment (Umeå University), the Artificial Leaf Project (K&A Wallenberg Foundation 2011.0055) and Energimyndigheten (36648-1). This research work used resources from the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported by the Office of Science, DOE, under contract no. DE-AC02-05CH11231. Testing of crystals and various parts of the setup were carried out at synchrotron facilities that were provided by the Advanced Light Source (ALS) in Berkeley and the Stanford Synchrotron Radiation Light Source (SSRL) in Stanford, which were funded by the DOE OBES. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH (grant P41GM103393). Use of the Linac Coherent Light Source (LCLS) and SSRL, SLAC National Accelerator Laboratory, is supported by the US DOE, Office of Science, OBES under contract no. DE-AC02-76SF00515. BNL's contribution to data collection at the LCLS is supported by the Life Science and Biomedical Technology Research (LSBR) program at the National Synchrotron Light Source II (NSLS-II), which operates under a DOE BER contract (DE-SC0012704) and DOE BES contract (DE-AC02-98CH10886), and is supported by NIH–NIGMS grant P41GM111244.

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A.M.O., J.K., V.K.Y. and J.Y. conceived the experiment; F.D.F., S.G., J.K., V.K.Y. and J.Y. designed the experiment; E.S.B., R.C., C.J.P., J.A.C., M.I., R.H., A.Z., H. Lebrette, V.S., M.Z., S.K., J.M., A.K.B., J.M.B., C.K., M.H., G.N.P. and R.D.V. prepared, characterized and provided the phytochrome, PS II and RNR samples; F.D.F., J.K., S.G., C.G.R. and A.M.O. designed the acoustic injectors; F.D.F., S.G., B.A., E.P., C.d.L., C.A.S., C.G.R., R.G.S., T.K., M.K., S.K., P.T.D., U.B., G.N.P., J.K., V.K.Y., A.M.O. and J.Y. performed the SFX and XES experiments; J.M.G., C.A.S., S.N., J.E.K., D.Z., M.C., S.S., H. Lemke, D.S., M.L. and R.A.-M. set up the beam lines; A.S.B., I.D.Y., T.M.-C., P.A., P.B., L.L., M.D.M., T.K., M. Amin, M. Allaire, F.D.F., J.K., E.S.B., T.F., C.W. and N.K.S. performed XRD and XES data analysis; F.D.F., J.K., E.S.B., A.M.O., V.K.Y. and J.Y. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Jan Kern or Vittal K Yachandra or Junko Yano.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Photograph of the DOT system and details of the optical pump setup.

a. A photograph of the drop-on-tape (DOT) device mounted at the X-ray Pump Probe (XPP) endstation of the Linac Coherent Light Source (LCLS) with various important components labeled. b. Schematic of the fiber optics setup for sample illumination. c. Example output of the feedback system to ensure that drop deposition is in phase with laser pump pulses. The arrival time of each drop over one of the IR “gates” is detected by changes in the IR transmission (yellow and pink traces). In addition, the laser transmission of the sample at each of the three illumination points on the tape for each individual drop is measured (blue and green traces shown here are for the pump #2 and #3) and delays are tuned to bring the IR and pump signals in phase. This information can also be used after the experiment to reject signal from drops that did not receive the correct illumination.

Supplementary Figure 2 Treatment of background originating from the Kapton belt.

a. Diffraction image showing the belt background on the XRD CCD detector with the maximum absorption of Kapton highlighted in red and the minimum in blue. b. A simplified geometry of the conveyor belt and the shadow it casts on the CCD. c. Illustrations of the parameters used in the Kapton absorption correction from left to right: on beam axis view, zoomed in beam axis view, and top view.

Supplementary Figure 3 Crystalline samples used in this study.

a. Crystal images of Phytochrome PAS-GAF region (~ 50 μm), b. Phytochrome PSM (~ 100 μm), c. RNR (20 - 30 μm), and d. PS II (20 - 50 μm).

Supplementary Figure 4 Statistics for three example diffraction experiments.

Shown is the indexing rate over time for suspensions of PAS-GAF (blue), PSM (red), PS II (turquoise) and RNR (black) crystals in the DOT setup. Data were collected at 10 Hz and the % of X-ray laser shots that yielded an indexable diffraction pattern are given as a function of total sample run time. Fluctuations in the indexing rate are largely a function of crystal density in the syringe, and also arise from the adjustment of the beam height in terms of the belt. Source data

Supplementary Figure 5 Bilin binding modes in DrBphP.

Four representative DrBphP structures were superposed. The positions of Asp207 and Tyr263 of the PSM (green, PDB code 4Q0J) and our room temperature, SFX PAS-GAF structure (yellow, PDB code 5MG0) were highly congruent, and indicate hydrogen bonding between the two residues. The positions of these residues differed from those found in a separate SFX structure collected at LCLS (magenta, PDB code 5L8M)1, and the photochemically compromised Asp207-Ala mutant (cyan, PDB code 4Q0I), which both show an outward splay of the Tyr263 side chain from residue 207, and absence of hydrogen bonding (e.g., a 3.9 Å inter-residue distance for 5L8M). The D-ring of our PAS-GAF structure appears to be in a position between that found for the PSM and the Asp207-Ala mutant. Pyrrole rings A and D are indicated.1. Edlund, P. et al. The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography. Sci. Rep. 6, 35279 (2016).

Supplementary Figure 6 Omit Fo-Fc electron density maps for the PAS-GAF and PSM constructs of DrBphP.

The two orthogonal views of the PAS-GAF maps (a and b) are calculated to 2.0 Å resolution and contoured at +3 σ (green) and -3 σ (red). Two orthogonal views of the PSM maps (c and d) calculated to 3.2 Å resolution and contoured at +3 σ (green) and -3 σ (red).

Supplementary Figure 7 XES data processing.

a. Raw image (minus outlier pixels), with wide ROI shown in red. b. Smooth polynomial background fit to data outside the wide ROI and extrapolated into the ROI (intensified by 4x for illustrative purposes). c. Integration of the wide ROI for both the raw and background. d. Corrected image with tight ROI based on Gaussian fit. e. Final corrected spectrum. Source data

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Fuller, F., Gul, S., Chatterjee, R. et al. Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers. Nat Methods 14, 443–449 (2017). https://doi.org/10.1038/nmeth.4195

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