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. 2017 Apr;14(4):443-449.
doi: 10.1038/nmeth.4195. Epub 2017 Feb 27.

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

Franklin D Fuller  1 , Sheraz Gul  1 , Ruchira Chatterjee  1 , E Sethe Burgie  2 , Iris D Young  1 , Hugo Lebrette  3 , Vivek Srinivas  3 , Aaron S Brewster  1 , Tara Michels-Clark  1 , Jonathan A Clinger  4 , Babak Andi  5 , Mohamed Ibrahim  6 , Ernest Pastor  1 , Casper de Lichtenberg  7 , Rana Hussein  6 , Christopher J Pollock  8 , Miao Zhang  6 , Claudiu A Stan  9 , Thomas Kroll  10 , Thomas Fransson  9 , Clemens Weninger  9   11 , Markus Kubin  12 , Pierre Aller  13 , Louise Lassalle  1 , Philipp Bräuer  13   14 , Mitchell D Miller  4 , Muhamed Amin  1 , Sergey Koroidov  7   9 , Christian G Roessler  5 , Marc Allaire  1 , Raymond G Sierra  11 , Peter T Docker  13 , James M Glownia  11 , Silke Nelson  11 , Jason E Koglin  11 , Diling Zhu  11 , Matthieu Chollet  11 , Sanghoon Song  11 , Henrik Lemke  11 , Mengning Liang  11 , Dimosthenis Sokaras  10 , Roberto Alonso-Mori  11 , Athina Zouni  6 , Johannes Messinger  7   15 , Uwe Bergmann  9 , Amie K Boal  8   16 , J Martin Bollinger Jr  8   16 , Carsten Krebs  8   16 , Martin Högbom  3   17 , George N Phillips Jr  4   18 , Richard D Vierstra  2 , Nicholas K Sauter  1 , Allen M Orville  13 , Jan Kern  1   11 , Vittal K Yachandra  1 , Junko Yano  1
Affiliations

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

Franklin D Fuller et al. Nat Methods. 2017 Apr.

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.

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Figures

Figure 1
Figure 1. Experimental Setup
a. A comparison of sample consumption rate (assuming 120 Hz operation) and pathlength accessible with the replenishing methods used at XFELs. b. The experimental setup of DOT. 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 cubic feet per hour purge flow. Droplets are deposited onto a polyimide belt using an ADE, and ejected from an open 2.5 mm diameter reservoir that is continuously resupplied via a capillary feed line attached to a syringe pump (top left b). In the interaction region, the belt was run at a small angle with respect to the z-axis (vertical) in the xz-plane (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 top right of panel b. Bottom right b. The XFEL beam passes parallel to the belt surface, striking the droplet atop the belt. c. 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.
Figure 2
Figure 2. Photo-initiated XES of PS II
a. The photoexcitation setup employed for PS II, comprised of a precision-machined grid of fixed excitation positions with 60 mm spacing, is shown. 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 Si-photodiodes for readout. b. A schematic of the mechanism depicting S-state advancement using periodic laser flashes. The interval between laser flashes 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 misses (see ref. 23). 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 using a freeze-quench method at 8 K with a synchrotron source (black). The XFEL spectra contain about 120,000 shots per difference spectrum. For details of the collection conditions and difference analysis see Online Methods and Supplementary Fig. 7.
Figure 3
Figure 3. XES of gas-activated RNR
a. A schematic of the differentially pumped oxygen gas activation setup is shown. b. The known reaction scheme of Ct RNR– is shown. Oxygen is activated at the MnII/FeII cluster to produce a high valent MnIVFeIV intermediate in a biomolecular reaction (kform = 13±3 mM−1sec−1 at 5 °C from the literature), which then decays to the active MnIVFeIII state (first-order kdecay = 0.02 sec−1 at 5 °C also from literature). 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 are computed via bootstrap residual sampling (1000 samples per data point). Difference spectra with respect to an 8 s exposure (smoothed by wavelet denoising) are shown in the main plot.
Figure 4
Figure 4. XRD of various enzymes
a. The PAS-GAF (blue and green, respectively) and the PSM (white) constructs from D. radiodurans BphP in their dark-adapted Pr states are shown superimposed. b. Superposition of atomic models of the PSM in the room-temperature Pr state (colored) with that derived from diffraction data collected at 100 K (white, PDB ID 4Q0J). The β-sheets of the GAF domains were superposed, 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 room temperature model the domains were colored blue, green, and orange for the PAS, GAF, and PHY domains, respectively. Several important features are labeled: HP, hairpin; NTE, amino-terminal extension; BV, biliverdin. BV from the room temperature structure is shown for orientation. c and d. Composite simulated-annealing omit maps (2Fo-Fc) contoured at 1 σ were superimposed with the corresponding PAS-GAF (c) or PSM (d) model of DrBphP. For clarity only electron density around Cys24 (gray), biliverdin (cyan) and for (c) the pyrrole water is shown. e. Metal site electron density (2Fo-Fc) at the heterodinuclear Mn/Fe site in aerobic class Ic RNR, metal ions and protein ligands are indicated (contoured to 1.3 σ) in blue. Residual positive difference electron density map (Fo-Fc) representing non-protein ligands is indicated in green (contoured to 3.5 σ). The manganese and iron 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.
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