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

Journal name:
Nature Methods
Volume:
14,
Pages:
443–449
Year published:
DOI:
doi:10.1038/nmeth.4195
Received
Accepted
Published online

Abstract

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.

At a glance

Figures

  1. Experimental setup.
    Figure 1: Experimental setup.

    (a) Qualitative comparison of sample-consumption rate (assuming 120 Hz operation) and path length accessible with the replenishing methods used at XFELs. (b) Schematic for DOT setup for experiments. The conveyor belt delivers droplets of sample at a high rate (up to 120 Hz). A large (283-liter) gas-tight chamber houses the entire instrument and is maintained at 95–97% helium via a 30 ft3/h purge flow. Droplets are deposited onto a polyimide belt by ADE from an open 2.5-mm-in-diameter reservoir that is continuously resupplied via a capillary feed line attached to a syringe pump (top left). In the interaction region, the belt was run at a small angle with respect to the z-axis (vertical) in the x,z plane (right; zoomed-in view of interaction point). Positioning the droplet in the X-ray focus is accomplished by moving the entire system in the horizontal plane, while the droplet z-position (vertical) on the tape at the intersection is adjusted by changing the deposition delay. The belt is cleaned and dried in situ, enabling continuous use for days. The reaction initiation point for longer time delays is shown in the green box. The XFEL beam passes parallel to the belt surface, striking the droplet, which is on the belt (right). (c) Schematic for the data collection geometry for XRD and XES. An inflatable (and X-ray transparent) plastic film door with a 160° aperture to the X-ray interaction region allows both XES and XRD to be collected simultaneously.

  2. Photo-initiated XES of PS II.
    Figure 2: Photo-initiated XES of PS II.

    (a) The photoexcitation setup used for PS II, which is comprised of a precision-machined grid of fixed excitation positions with 60-mm spacing. Optical gates, which measure droplet arrival times on the grid, are realized by two low-power (<0.5 mW) continuous-wave near-infrared (NIR, 850 nm) point sources, delivered via optical fiber. NIR light scatters from the droplets as they pass over the gates and is collected onto high-speed silicon photodiodes for readout. (b) Schematic of the mechanism depicting S-state advancement using periodic laser flashes. The interval between laser flashes (ΔT) for both DOT and freeze–quench methods was 1.0 s. Flash states (denoted as 0F, 1F, 2F or 3F) are highly enriched in the pure reaction intermediates S1, S2, S3 and S0, respectively, but are not completely pure due to small back-reaction rates and photon misses23. (c) A comparison of Mn Kβ1,3 XES difference spectra of PS II solution collected with the DOT instrument at room temperature using an XFEL (red) and the same state differences collected by using a freeze–quench method at a temperature of 8 K with a synchrotron source (black). The XFEL spectra contain about 120,000 shots per difference spectrum. a.u., arbitrary units. For details of the collection conditions and difference analysis, see Online Methods and Supplementary Figure 7.

  3. XES of gas-activated RNR.
    Figure 3: XES of gas-activated RNR.

    (a) Schematic of the differentially pumped O2 gas activation setup containing regions of O2 gas and slight negative pressure (vacuum). (b) The known reaction scheme of Ct RNR17, 18, 19 is shown. O2 is activated at the Mn(II)–Fe(II) cluster to produce a high-valent Mn(IV)–Fe(IV) intermediate in a biomolecular reaction (kform = 13 ± 3 mM−1s−1 at 5 °C from the literature18), which then decays to the active Mn(IV)–Fe(III) state (first-order kdecay = 0.02 s−1 at 5 °C also from the literature18). (c) RNR solution was monitored at 25 °C with Fe Kα emission for various O2 exposure times. The inset shows the Kα1 FWHM as a function of exposure time relative to an 8 s exposure. Error bars were computed via bootstrap residual sampling (1,000 samples per data point). Difference spectra with respect to an 8 s exposure (smoothed by wavelet denoising and 10× magnification) are shown in the main plot.

  4. XRD of various enzymes.
    Figure 4: XRD of various enzymes.

    (a) Structures of the PAS–GAF (blue and green, respectively) and PSM (white) domains from D. radiodurans BphP in their dark-adapted Pr states are shown superimposed. HP, hairpin; NTE, amino-terminal extension; BV, biliverdin. (b) Superposition of atomic models of the PSM in the room-temperature Pr state (colored) with that derived from diffraction data collected at a temperature of 100 K (white; PDB ID 4Q0J). The β-sheets of the GAF domains were superimposed, allowing the respective positions of the PAS and PHY domains of the two models to be identified. The largest differences between the models were found at the PHY domain. For the model derived from the data collected at room temperature, the domains were colored blue, green and orange for the PAS, GAF and PHY domains, respectively. BV from the room temperature structure is shown for orientation. (c,d) Composite simulated-annealing omit maps (2FoFc) contoured at 1 σ were superimposed with the corresponding PAS–GAF (c) or PSM (d) model of DrBphP22. For clarity, only the electron density around Cys24 (gray; c,d), biliverdin (cyan; c,d) and the pyrrole water (c, red sphere) is shown. Labels A and D indicate the first and fourth pyrrole rings of the BV chromophore, respectively. (e) Metal-site electron density (2FoFc) at the heterodinuclear Mn–Fe site in an aerobic class Ic RNR, metal ions and protein ligands are shown (contoured to 1.3 σ) in blue. Residual positive-difference electron density (FoFc), which represents non-protein ligands, is shown in green (contoured to 3.5 σ). The Mn and Fe atoms are depicted as purple and orange spheres, respectively. Kβ1,3 XES of oxidized RNR in crystal and in solution collected at room temperature with an XFEL is shown to the left. A Mn(II)Cl2 calibration standard is also shown to illustrate the absolute oxidation state of the solution and crystal spectra. Fe Kα XES data collected from oxidized RNR crystals is shown at the right.

  5. Photograph of the DOT system and details of the optical pump setup.
    Supplementary Fig. 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.

  6. Treatment of background originating from the Kapton belt.
    Supplementary Fig. 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.

  7. Crystalline samples used in this study.
    Supplementary Fig. 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).

  8. Statistics for three example diffraction experiments.
    Supplementary Fig. 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.

  9. Bilin binding modes in DrBphP.
    Supplementary Fig. 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).

  10. Omit Fo-Fc electron density maps for the PAS-GAF and PSM constructs of DrBphP.
    Supplementary Fig. 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).

  11. XES data processing.
    Supplementary Fig. 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.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

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Author information

  1. Present addresses: Center for Photonics and Smart Materials, Zewail City of Science and Technology, Giza, Egypt (M.A.), Ventana Medical Systems, Inc., Tucson, Arizona, USA (C.G.R.), and SwissFEL, Paul Scherrer Institut, Villigen, Switzerland (H. Lemke).

    • Muhamed Amin,
    • Christian G Roessler &
    • Henrik Lemke
  2. These authors contributed equally to this work.

    • Franklin D Fuller &
    • Sheraz Gul

Affiliations

  1. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

    • Franklin D Fuller,
    • Sheraz Gul,
    • Ruchira Chatterjee,
    • Iris D Young,
    • Aaron S Brewster,
    • Tara Michels-Clark,
    • Ernest Pastor,
    • Louise Lassalle,
    • Muhamed Amin,
    • Marc Allaire,
    • Nicholas K Sauter,
    • Jan Kern,
    • Vittal K Yachandra &
    • Junko Yano
  2. Department of Biology, Washington University in St. Louis, St. Louis, Missouri, USA.

    • E Sethe Burgie &
    • Richard D Vierstra
  3. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

    • Hugo Lebrette,
    • Vivek Srinivas &
    • Martin Högbom
  4. Department of BioSciences, Rice University, Houston, Texas, USA.

    • Jonathan A Clinger,
    • Mitchell D Miller &
    • George N Phillips Jr
  5. National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, New York, USA.

    • Babak Andi &
    • Christian G Roessler
  6. Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany.

    • Mohamed Ibrahim,
    • Rana Hussein,
    • Miao Zhang &
    • Athina Zouni
  7. Institutionen för Kemi, Kemiskt Biologiskt Centrum, Umeå Universitet, Umeå, Sweden.

    • Casper de Lichtenberg,
    • Sergey Koroidov &
    • Johannes Messinger
  8. Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA.

    • Christopher J Pollock,
    • Amie K Boal,
    • J Martin Bollinger Jr &
    • Carsten Krebs
  9. Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, California, USA.

    • Claudiu A Stan,
    • Thomas Fransson,
    • Clemens Weninger,
    • Sergey Koroidov &
    • Uwe Bergmann
  10. SSRL, SLAC National Accelerator Laboratory, Menlo Park, California, USA.

    • Thomas Kroll &
    • Dimosthenis Sokaras
  11. LCLS, SLAC National Accelerator Laboratory, Menlo Park, California, USA.

    • Clemens Weninger,
    • Raymond G Sierra,
    • James M Glownia,
    • Silke Nelson,
    • Jason E Koglin,
    • Diling Zhu,
    • Matthieu Chollet,
    • Sanghoon Song,
    • Henrik Lemke,
    • Mengning Liang,
    • Roberto Alonso-Mori &
    • Jan Kern
  12. Institute for Methods and Instrumentation on Synchrotron Radiation Research, Helmholtz Zentrum Berlin für Materialien und Energie GmbH, Berlin, Germany.

    • Markus Kubin
  13. Diamond Light Source Limited, Harwell Science and Innovation Campus, Didcot, UK.

    • Pierre Aller,
    • Philipp Bräuer,
    • Peter T Docker &
    • Allen M Orville
  14. Department of Biochemistry, University of Oxford, Oxford, UK.

    • Philipp Bräuer
  15. Department of Chemistry–Ångström, Molecular Biomimetics, Uppsala University, Uppsala, Sweden.

    • Johannes Messinger
  16. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA.

    • Amie K Boal,
    • J Martin Bollinger Jr &
    • Carsten Krebs
  17. Department of Chemistry, Stanford University, Stanford, California, USA.

    • Martin Högbom
  18. Department of Chemistry, Rice University, Houston, Texas, USA.

    • George N Phillips Jr

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Photograph of the DOT system and details of the optical pump setup. (614 KB)

    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.

  2. Supplementary Figure 2: Treatment of background originating from the Kapton belt. (196 KB)

    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.

  3. Supplementary Figure 3: Crystalline samples used in this study. (563 KB)

    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).

  4. Supplementary Figure 4: Statistics for three example diffraction experiments. (208 KB)

    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.

  5. Supplementary Figure 5: Bilin binding modes in DrBphP. (193 KB)

    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).

  6. Supplementary Figure 6: Omit Fo-Fc electron density maps for the PAS-GAF and PSM constructs of DrBphP. (531 KB)

    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).

  7. Supplementary Figure 7: XES data processing. (549 KB)

    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.

PDF files

  1. Supplementary Text and Figures (1,388 KB)

    Supplementary Figures 1–7 and Supplementary Tables 1–2

  2. Supplementary Protocol (3,443 KB)

    Supplementary Protocol

Additional data