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
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- 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.
- 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.
- 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).
- 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.
- 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).
- 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).
- 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.