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Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

Abstract

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

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Figure 1: The concentric-flow MESH injector setup at the CXI instrument of the LCLS.
Figure 2: Comparison of cryo-cooled and ambient-temperature T. thermophilus 30S-paromomycin complex structures.
Figure 3: Structural changes were observed in h28 and in paromomycin binding at ambient temperature.

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Acknowledgements

Portions of this research were carried out at the LCLS at the SLAC National Accelerator Laboratory. The LCLS is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences (OBES), under contract DE-AC02-76SF00515. This research used resources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported by the Office of Science, DOE, under contract DE-AC02-05CH11231. Part of the sample-injector mechanical system used at LCLS for this research was funded by the US National Institutes of Health (NIH) (P41GM103393, formerly P41RR001209). J.Y. and V.K.Y. are supported by the Office of Science, OBES, Chemical Sciences, Geosciences, and Biosciences (CSGB) of the DOE under contract DE-AC02-05CH11231 for X-ray methodology and instrumentation. The LCLS is acknowledged for beam time access under experiments cxig7014, cxib6714 and cxig3614. E.H.D., H.L., R.G.S., M.J.B., C.Y.H., C.A.S. and H.D. acknowledge the support of the OBES through the AMOS program within the CSGB and of the DOE through the SLAC Laboratory Directed Research and Development Program. N.K.S. acknowledges an LBNL Laboratory Directed Research and Development award under contract DE-AC02-05CH11231. E.H.D. acknowledges financial support from the Stanford University Dean of Research. H.D., S.M.S. and J.D.P. acknowledge support from the joint Stanford ChEM-H and SLAC National Accelerator Laboratory seed grant program. This work is supported by NIH grants GM51266 (to J.D.P.), GM082545 (to E.V.P.), GM055302 (to V.K.Y.), GM110501 (to J.Y.), GM095887 and GM102520 (to N.K.S.); the DFG–Cluster of Excellence “UniCat” coordinated by the Technische Universitaet at Berlin and Sfb1078, TP A5 (to A.Z. and M.I.); and Human Frontiers Science Project awards RGP0005/2011 (to H.L.) and RGP0063/2013 310 (to J.Y. and A.Z.). C.G. kindly thanks the PIER Helmholtz Graduate School, as well as the Helmholtz Association for financial support. H.D. acknowledges valuable discussions with A. Takeuchi, K. Dursuncan and E. Satunaz. We thank M. West for support in designing and machining the injector load lock setup and G. Stewart for excellent technical assistance with creating the graphics for Figure 1.

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Authors and Affiliations

Authors

Contributions

H.D. and R.G.S. designed and coordinated the project. R.G.S., H.L., H.D., C.A.S., C.Y.H., S.G., F.F. and M.J.B. developed the coMESH injection method. C.G. executed the ribosome data reduction. H.D. refined ribosome and thermolysin structures. N.K.S., A.S.B., I.D.Y. and T.M.-C. processed PSII and thermolysin diffraction data. H.D., E.H.D., B.H., R.C., I.D.Y., M.I., J.K. and A.Z. prepared samples. R.G.S., H.D., H.L., C.A.S., E.H.D., S.G., F.F., J.K., R.C., M.I., A.A., M.L., M.S.H., J.E.K., S.B., E.A.J., J.Y. and V.K.Y. carried out the experiment. H.D., C.G., R.G.S., E.V.P., J.K., J.Y., V.K.Y., S.M.S. and J.D.P. analyzed data. H.D., R.G.S., E.H.D., C.G., H.L., C.A.S. and J.K. prepared the manuscript with input from all other coauthors.

Corresponding authors

Correspondence to Raymond G Sierra or Hasan DeMirci.

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Competing interests

R.G.S., H.L. and M.J.B. have applied for a patent (US Patent Application 13/896,303) describing the MESH and coMESH designs.

Integrated supplementary information

Supplementary Figure 1 A reference image of a capillary not flowing any liquid and with minimal buildup on the outer surface.

This configuration is typical of the protruded coMESH. The co-terminal MESH would have the smaller inner capillary obstructed from view by the larger outer sheath capillary.

Supplementary Figure 2 A picture of the 360-μm cross fitting used to create the mixing manifold for the coMESH system.

The northern channel contains the 100 μm × 160 μm × 1.5 m fused silica capillary, 1/16” (1.6 mm) FEP tubing sleeve, and appropriate 1/16” (1.6 mm) to 360 μm adapter fittings. The capillary passes through the cross and ends at the tip of the south channel seen in the bottom of the figure (not visible, obstructed). Coterminal with the main sample line, is the outer sheath capillary, 200 μm × 360 μm × 5 cm, seen in the south. The east line is a 75 μm × 360 μm × 1 m capillary and the appropriate 360 μm fitting. The west channel is sealed with a plug fitting.

Supplementary Figure 3 coMESH injection of 30S ribosomal subunit, PSII and thermolysin crystals.

The scale bars in each picture are 360 μm. (a) 30S ribosomal subunit crystals: 17% (v/v) MPD mother liquor in 100 μm ID capillary. The sheath flow capillary is 75 μm ID,running 34% (v/v) MPD sister liquor. The outer concentric capillary is 200 μm ID × 5 cm long tapered fused silica capillary coterminal with the inner capillary. The sheath flow was charged by a charging union at 3,000 V and the counter electrode was grounded. The sample ran at 1.5 μl/min for almost an hour, hence the buildup evident from debris created by the intense X-ray interaction. (b) PSII crystals (same capillary conditions as (a)): with the appropriate PSII mother and sister liquor described. Here the sample flow was charged by the syringe reservoir’s needle to 7,000 V with the counter electrode at ground. The syringe pump was set to 1 μl/min. (c) PSII crystals in modified protruded configuration: Here the flow parameters and geometries are the same as in (b), with the exception that the outer line is simply primed and then capped off and the outer concentric capillary is recessed about 1 mm. (d) Thermolysin crystal standard in modified protruded coMESH: similar flow conditions to (c) with samples in 38% (v/v) glycerol, 50 mM CaCl2 and 0.1M MES. The speckles in the capillary are crystals flowing towards the interaction region.

Supplementary Figure 4 coMESH injection of PSII solution.

(a) PSII dimer solution (non-crystalline): 6.5 mM chlorophyll, in 40% (v/v) glycerol, 100 μm ID × 160 μm OD × 1.5 m long fused silica capillary. The sheath flow capillary was a 30 μm ID × 150 μm OD × 1 m fused silica capillary, primed with 40% (v/v) glycerol, 0.1 M MES pH 6.5, 5 mM CaCl2. After priming, the atmospheric side of the line was capped off. The outer concentric capillary seen in the figure was a 180 μm ID × 360 μm OD × 5 cm long, tapered fused silica capillary. The sample flow was charged by the needle of the reservoir syringe and was pushed by a syringe pump set to 2 μl/min. The running voltage was 6,000 V and the counter electrode was grounded. (b) A higher resolution image of a PSII solution (non-crystalline) in the protruding coMESH configuration, to emphasize the presence of a wavy irregular appearance at the capillary outer wall, which was possibly a thin coating of liquid from the sheath to aid the meniscus of the sample capillary. (c) A picture of an injector failure in a single capillary (top of the figure behind the scale bar) configuration (large black build-up is frozen PEG-solution at the injector tip and red background is from illumination LED). The solution was a high molecular weight PEG solution. This solution failed in a very similar manner when unassisted by a sister liquor in an extended coMESH configuration.

Supplementary Figure 5 Diffraction images of PSII microcrystals measured with two MESH configurations.

Images are composite powder patterns, showing the maximum pixel values observed over all diffraction events during 9 minute experimental runs for PSII crystals in (a) the protruded coMESH configuration aimed to minimize background scatter, however, it created diffraction rings from fluid freezing and precipitation (b) the co-terminal coMESH configuration. The crystals were suspended in 0.1 M TRIS, 0.1 M ammonium sulfate, 5% (v/v) ethylene glycol, 35% (w/v) PEG 5000. The sheath flow in (b) was 50% (v/v) ethylene glycol, 50 mM TRIS, and 50 mM ammonium sulfate. In condition (a), using the single-capillary MESH configuration without sheath showed evidence of fluid freezing and precipitation (indicated by arrows at 3.47, 2.70 and 2.25 Å). Resolution is indicated by orange rings (4.0 and 2.8 Å(a) and 4.0, 2.8 and 2.5 Å (b)) for reference.

Supplementary Figure 6 Electron-density map of thermolysin obtained by using microcrystals injected by coMESH.

Close-up view of a representative helix structure residues Ser65 (bottom) - His88 (top) with calculated 2Fo – Fc electron density map contoured at the 1.0 σ level.

Supplementary Figure 7 Schematic of the load lock system used for positioning the injector capillary close to the interaction region.

In its operating position (a) the capillary tip is located close to the interaction region (x) and can be precisely positioned by an XYZ stage (not shown). The coMESH setup is represented by the yellow block with connecting capillary lines that pass through the 6” (15.24 cm) flange out of vacuum. In order to clean or change the capillary the load lock bellows can be extended and after closing the gate valve on top of the experimental chamber, can be tilted by 90° (b) to allow easy access to the injector capillary. The entire injector assembly is comprised of the feedthrough flange and mounting rod (black), the counter electrode (gray trapezoid) and PEEK holder (brown). The assembly is mounted to a 6” (15.24 cm) flange at the top of the load lock bellows and can be easily swapped for a second injector assembly if necessary. The flange has all the necessary feed-throughs for the liquid lines, high voltage connections and three optical fiber feed-throughs for optional in situ sample illumination.

Supplementary Figure 8 A photo of the coMESH setup running ribosome crystals at the CXI beamline, prior to receiving X-rays.

On the left, the custom loadlock (A) is mounted on the top of the main CXI (1 × 1 μm2 beam) sample chamber (not visible); the stick is lowered allowing the coMESH to reach the interaction region. Both capillaries and grounding line are attached to appropriate feedthroughs at the top of the 6” (15.24 cm) flange of the sample stick (B). Not seen is the inline charging fitting connected to the purple polymer tubing (C) feeding the sister liquor. On the right, the high voltage supply (D) is seen in front of the sample rocker (E) holding the custom sample reservoir, with the syringe pump and sister liquor syringe reservoir seen further back (F).

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Supplementary Figures 1–8 and Supplementary Tables 1–3 (PDF 1059 kb)

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Sierra, R., Gati, C., Laksmono, H. et al. Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II. Nat Methods 13, 59–62 (2016). https://doi.org/10.1038/nmeth.3667

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