High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions

Abstract

Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.

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Figure 1: Mylar in situ double-sandwich plates.
Figure 2: A flowchart summarizing the steps involved in setting up protein crystallization trials for data collection and screening purposes.
Figure 3: 3D-printed holders suitable for differently sized patches of in situ film sandwiches.
Figure 4: Design features of G-holders.
Figure 5: Design features of GT-holders.
Figure 6: Design features of GAT-holders.
Figure 7: Customized in situ plates.
Figure 8: Overview of customizable design features for the Mylar in situ method.
Figure 9: Overview of pipetting patterns on in situ crystallization plates.
Figure 10: Software windows of the 3D design process.
Figure 11: Crystal harvesting from in situ crystallization plates.
Figure 12: Structures solved by the Mylar in situ method.
Figure 13: Schematics for vapor-diffusion crystallization on in situ plates, and for adding cryoprotectants post crystal growth.

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Acknowledgements

We thank members of the Ernst group, in particular A.R. Balo and Y. Shen, for their contributions to the original work. We acknowledge the MADLab and the Gerstein Library, mainly E. Lenton and M. Spears (University of Toronto), for admission to the 3D-printing facility. We also thank A. Trnka (Saunders) for supplying us with various spacers. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We specifically thank the staff at the GM/CA beamline. Videos of the Irelec CATS robot mounting 1-well holders were shot in the LS-CAT ID-D endstation, Sector 21, at the APS, for which we acknowledge J. Brunzelle, a member of the LS-CAT staff. S. Haider (Formulatrix) kindly calibrated a Mylar in situ plate for use with the Rock Imager. For fruitful discussions and carefully reading the manuscript, we are grateful to E.F. Pai (University of Toronto) and S. Keller (University of Kaiserslautern). This work was supported by a Research Fellowship from the German Research Foundation (DFG) to J.B. (BR 5124/1-1), by National Institutes of Health grant R01 GM108635 (to V.C.), and by funding from the Canadian Institute for Advanced Research (CIFAR; to O.P.E.). O.P.E. holds a Canada Excellence Research Chair Award and the Anne and Max Tanenbaum Chair in Neuroscience at the University of Toronto.

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Contributions

J.B. and O.P.E. designed the research. J.B. wrote the manuscript. All authors commented on the manuscript. V.C. supervised A2A work. J.B., T.M., W.-L.O., A.I., and M.-Y.L. grew crystals, and collected and analyzed the data. V.K. helped with holder designs. A.K. grew SWMb crystals. D.J.K. and C.M.O. helped with data collection and implementation of the Mylar in situ setup at the APS synchrotron. S.X. and O.M. designed the GM/CA adaptors, including the translation stages.

Corresponding authors

Correspondence to Jana Broecker or Oliver P Ernst.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Crystals grown on or transferred to in situ plates.

(a) HEWL crystals (PDB IDs 5KKI and 5KKJ). (c) SWMb crystals (PDB IDs 5KKK and 5WJK). (d) HwBR crystals (PDB ID 5KKH). (e) A2AAR crystals (PDB ID 5VRA). (f) Opsin crystals (PDB ID 5WKT). (b) Diffraction images for HEWL crystals of one and the same in situ well (left) at the beginning of data collection at room temperature (detector distance: 350 nm) as well as (right) after 3 h into data collection (detector distance: 300 nm). High-resolution diffraction spots at 1.9 Å are indicated. Scale bars are (a, d–f) 100 μm and (c) 200 μm, respectively.

Supplementary Figure 2 Lining a glass plate with Mylar film.

(a) Arrangement of materials on the bench (Step 1). (b) Securing Mylar film on one side of the glass plate (Step 2). (c–d) Lining the glass plate with Mylar film, cutting it using a pair of scissors, and wrapping it around the bottom edge (see Steps 3–4). (e) Smoothening the Mylar film onto the glass plate (Step 5). (f) Mylar-lined glass plate either to be used as an in situ cover plate or ready for attaching spacer tape, when preparing an in situ base plate.

Supplementary Figure 3 Preparing and loading the in situ base plate as well as forming and sealing the inner film sandwich.

(a) Placing spacer tape onto a Mylar-lined glass plate (Step 8). (b) Folding over the bottom right corner of the spacer’s brown protective tape (Step 9). (c) Setting up crystallization trials (see Steps 10–12 and Box 3), here using a crystallization robot. (d) Cutting the Mylar film on the outside of the freshly formed double sandwich glass plate (Step 16). (e) Removing Mylar overhangs from the exposed inner film sandwich (Step 19). (f) Sealing the glass sandwich with layers of nail polish (Steps 22–23).

Supplementary Figure 4 Opening in situ plates and loading wells onto holders.

(a) Cutting the outer seal (Steps 31–32). (b) Opening the glass sandwich by lifting off the top glass plate (Steps 33–34). The positions of the film sandwich (green area) and of well A1 (black arrow) are indicated. (c) Cutting out wells using a fresh razor blade (Step 36). (d) Isolating wells to be loaded onto a holder using fine-point tweezers (Step 37). (e) Loading a well into a tweezer holder (Step 38.Ai). (f) Applying a layer of glue onto GT- or GAT-holders (Step 38.Ci). (g) Attaching an array of wells onto a sticky GT- or GAT-holder (Steps 38.Cii–iii).

Supplementary Figure 5 Handling in situ wells at the beam line.

(a) At room temperature, crystals (colored on the left, colorless on the right) are usually visible by eye. However, under cryogenic conditions, when the mesophase turns turbid (b), it can be difficult to spot crystals particularly those that are not as colorful as those shown in (c). (d) It is helpful to take a snapshot of the well prior to mounting the particular holder in order to better locate crystals for data collection. Snapshots in a–c were taken through the on-axis camera at the beam line. The snapshot in d was taken at a synchrotron through the ocular of a microscope using a smartphone. Scale bars are (a) 20 μm and (b–d) 4 mm, respectively.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1 and 2, the Supplementary Methods, and Supplementary Manuals 1 and 2. (PDF 1653 kb)

Supplementary Data 1–4

Supplementary Data 1. Printer files for 1-well G-holders. Supplementary Data 2. Printer files for 4-well G-holders. Supplementary Data 3. Printer files for GT-holders. Supplementary Data 4. Printer files for GAT-holders. (ZIP 2180 kb)

GM/CA robot. Utomounter robot at GM/CA mounting and demounting 1-well holders stored in liquid nitrogen. (MP4 9940 kb)

Irelec CATS robot. Commercially available Irelec CATS robot mounting and demounting 1-well holders stored in sample vials in liquid nitrogen. (MP4 24590 kb)

Tweezer holder. Loading an individual well to a tweezer holder (1-well G-holder). (MP4 7206 kb)

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Broecker, J., Morizumi, T., Ou, W. et al. High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions. Nat Protoc 13, 260–292 (2018). https://doi.org/10.1038/nprot.2017.135

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