9p16
Thermolysin Room-Temperature In-Situ, Grown On-Site
Structural highlights
FunctionTHER_GEOSE Extracellular zinc metalloprotease.[1] Publication Abstract from PubMedRoom-temperature (RT) X-ray diffraction experiments enable us to investigate protein dynamics, efficiently probe fragment binding, and perform 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 RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals, improving crystal integrity for fragile crystals. While many X-ray sources are now equipped to grow crystals on site for in-situ experiments, to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ambient temperature for RT data collection, while the equivalent methodology for cryo-cooled crystals is well established. Here we examine the impact of shipping on crystals grown on MiTeGen In Situ-1 plates at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI) in Buffalo, NY, United States transatlantic to DLS in Didcot, United Kingdom. We utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Box Thermal Shipper (Blue Box), which can maintain temperature for up to 168 hours, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping 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, 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. Does crossing the pond affect crystal quality?,Campomizzi CS, Snell ME, Mikolajek H, Sandy J, Sanchez-Weatherby J, Budziszewski GR, Russi S, Howells R Jr, Cohen A, Hough MA, Bowman SEJ bioRxiv [Preprint]. 2025 Jun 17:2025.06.12.659325. doi: , 10.1101/2025.06.12.659325. PMID:40666996[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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