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bioRxiv · 10.1101/2025.06.12.659325

Does crossing the pond affect crystal quality?

Campomizzi, C. S.·Snell, M. E.·Mikolajek, H.·Sandy, J.·Sanchez-Weatherby, J.·Budziszewski, G. R.·Russi, S.·Howells, R.·Cohen, A.·Hough, M. A.·Bowman, S. E. J.
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Abstract

Room-temperature X-ray diffraction experiments can facilitate investigation of protein dynamics, efficient probing of fragment binding, and time-resolved crystallography experiments. The Versatile Macromolecular Crystallography in situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the collection of room-temperature X-ray diffraction data in situ directly from crystallization plates without any manipulation of protein crystals, preserving the integrity of fragile samples. While many X-ray light sources are now equipped to grow crystals on site for room-temperature experiments, to date there has been no comprehensive analysis of the effect of shipping crystals within plates at ambient temperature for in situ room-temperature data collection. In situ methods place more stringent demands on shipping, as crystallization drops must arrive both intact and properly positioned within the plate to remain in the field of view of crystal imaging systems. The latter requirement imposes more stringent limits on shipping procedures as lateral movement of <1mm may leave a droplet out of the crystal imager field of view yet is independent of the requirement for the diffraction quality of crystals to be maintained. In contrast, the equivalent methodology for cryo-cooled crystals is well established. Here we investigate the impact of transatlantic shipping of crystals generated from commercially available proteins (lysozyme, thaumatin, and thermolysin) within the same plates in which they were grown. MiTeGen In Situ-1 plates containing the protein crystals were set up at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI, Buffalo, NY, United States) and shipped to DLS (Didcot, United Kingdom). Identical plates were prepared on site at DLS in the Crystallisation Facility at Harwell. We also performed this experiment with human lysine acetyltransferase 2B (KAT2B) to validate our proposed pipeline with a typical protein crystal sample. For shipping, we utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Thermal Shipping Box (Blue Box), which can maintain temperature for a few days, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping by standard commercial courier might compromise data quality through mechanical stress or temperature fluctuations. Instead, we found that room-temperature data collected at VMXi showed no significant differences for crystals set up at UB-HWI and shipped relative to crystals set up on site in the UK. High-resolution structures were successfully determined for all proteins in the study (both those shipped from UB-HWI and set up on-site at DLS), demonstrating that long-distance shipment of crystals at non-cryogenic temperatures is feasible without compromising diffraction quality. This study provides a proof-of-concept workflow for expanding access to room-temperature crystallography worldwide, enabling more researchers to leverage cutting-edge techniques without needing to grow crystals on site. SynopsisThe effect of international shipment of protein crystals on in situ room-temperature X-ray diffraction quality.

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Campomizzi, C. S., Snell, M. E., Mikolajek, H., Sandy, J., Sanchez-Weatherby, J., Budziszewski, G. R., Russi, S., Howells, R., Cohen, A., Hough, M. A., Bowman, S. E. J.. 2025-06-17. Does crossing the pond affect crystal quality?. https://doi.org/10.1101/2025.06.12.659325

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