Does crossing the pond affect crystal quality?

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View ORCID Profile Christopher S. Campomizzi , View ORCID Profile M. Elizabeth Snell , View ORCID Profile Halina Mikolajek , View ORCID Profile James Sandy , View ORCID Profile Juan Sanchez-Weatherby , View ORCID Profile Gabrielle R. Budziszewski , View ORCID Profile Silvia Russi , Richard Howells Jr , View ORCID Profile Aina Cohen , View ORCID Profile Michael A. Hough , View ORCID Profile Sarah E.J. Bowman doi: https://doi.org/10.1101/2025.06.12.659325 Christopher S. Campomizzi a University at Buffalo Hauptman Woodward Institute , 700 Ellicott Street, Buffalo, NY, 14203, United States b Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, SUNY Buffalo , 955 Main Street, Buffalo, NY, 14203, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christopher S. Campomizzi M. Elizabeth Snell a University at Buffalo Hauptman Woodward Institute , 700 Ellicott Street, Buffalo, NY, 14203, United States b Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, SUNY Buffalo , 955 Main Street, Buffalo, NY, 14203, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for M. Elizabeth Snell Halina Mikolajek c Diamond Light Source Ltd , Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom d The Research Complex at Harwell , Harwell Science and Innovation Campus, Didcot, OX11 0FA, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Halina Mikolajek James Sandy c Diamond Light Source Ltd , Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for James Sandy Juan Sanchez-Weatherby c Diamond Light Source Ltd , Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Juan Sanchez-Weatherby Gabrielle R. Budziszewski a University at Buffalo Hauptman Woodward Institute , 700 Ellicott Street, Buffalo, NY, 14203, United States b Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, SUNY Buffalo , 955 Main Street, Buffalo, NY, 14203, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabrielle R. Budziszewski Silvia Russi e Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University , Menlo Park, CA, 94025, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Silvia Russi Richard Howells Jr f Crystal Positioning Systems , 1070 Allen Street, Jamestown, NY, 14701, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aina Cohen e Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University , Menlo Park, CA, 94025, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aina Cohen For correspondence: acohen{at}slac.stanford.edu michael.hough{at}diamond.ac.uk sebowman{at}buffalo.edu Michael A. Hough c Diamond Light Source Ltd , Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom d The Research Complex at Harwell , Harwell Science and Innovation Campus, Didcot, OX11 0FA, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael A. Hough For correspondence: acohen{at}slac.stanford.edu michael.hough{at}diamond.ac.uk sebowman{at}buffalo.edu Sarah E.J. Bowman a University at Buffalo Hauptman Woodward Institute , 700 Ellicott Street, Buffalo, NY, 14203, United States b Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, SUNY Buffalo , 955 Main Street, Buffalo, NY, 14203, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sarah E.J. Bowman For correspondence: acohen{at}slac.stanford.edu michael.hough{at}diamond.ac.uk sebowman{at}buffalo.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Room-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. 1. Introduction X-ray crystallography remains the predominant technique for determination of three-dimensional biomolecular structures, accounting for over 80% of structures deposited into the Protein Data Bank ( https://www.rcsb.org/stats/summary ) ( Berman et al ., 2000 ). While methods such as cryo-electron microscopy are becoming more prevalent for structure determination, diffraction-based methods are currently undergoing an explosion of technique development ( Bjelcic et al ., 2023 , Ebrahim et al ., 2022 , Foos et al ., 2024 , Khusainov et al ., 2024 , Mikolajek et al ., 2023 , Moreno-Chicano et al ., 2022 , Okumura et al ., 2022 ) enabled by advances in X-ray sources and beamline optics ( Chapman et al ., 2011 , Eriksson et al ., 2014 , Hegde et al ., 2025 , McNeil & Thompson, 2010 , Schneider et al ., 2021 , Sierra et al ., 2019 , Warren et al ., 2024 ), as well as detector technology ( Dinapoli et al ., 2011 , Hatsui & Graafsma, 2015 , Mozzanica et al ., 2018 , Tolstikova et al ., 2019 , van Driel et al ., 2020 ). An overwhelming majority of structures from X-ray crystallography have been determined at cryogenic (cryo) temperature. Cryo-crystallography has contributed extensively to structural biology, offering several practical advantages, including greatly increasing the absorbed X-ray dose that a sample can withstand without excessive loss of diffraction due to radiation damage ( Young et al ., 1990 ), as well as ease of transport and compatibility with remote data collection ( Soltis et al ., 2008 ). Despite these advantages, there are limitations in using cryo-cooled crystals. First, cryo-cooling a crystal can ‘trap’ the macromolecule in a non-biologically relevant protein conformation. Second, protein crystals are often delicate, making them difficult to harvest and cryopreserve ( Teng, 1990 ). Third, structures from diffraction data collected at cryogenic temperatures below the glass transition are snapshots of a particular state of a protein; dynamics and conformational fluctuations are not typically observable ( Juers & Matthews, 2001 , Keedy, Fraser, et al ., 2015 , Keedy et al ., 2014 ). Room-temperature (RT) crystallography is re-emerging as a valuable approach, offering insight into structural data that aligns better with data collected from solution-based methods, and in many cases these data are more biologically relevant ( Fenwick et al ., 2014 , Fraser et al ., 2009 , Keedy, Kenner, et al ., 2015 ). Further, there is no need for addition of cryoprotectants, which may disrupt local structure and ligand binding sites. Notably, the field of time resolved serial crystallography at X-ray Free Electron Laser sources and synchrotron beam lines is almost entirely dependent on room-temperature crystallographic experiments ( Chapman et al ., 2011 , Gati et al ., 2014 ). Despite its potential, RT crystallography presents several key challenges, particularly in crystal transportation and handling. While shipping of dewars containing cryo-cooled crystals has become routine with most synchrotron users using this approach together with remote access control of X-ray beamlines, equivalent approaches for RT crystals are far less well developed. One major challenge is keeping crystals hydrated during transportation and sample handling at the beamline. Originally, crystals were mounted in glass capillaries ( Blundell & Johnson, 1976 ), but recently the use of plastic capillaries ( Kalinin et al ., 2005 ) or hydration streams at beamlines ( Russi et al ., 2011 , Sanchez-Weatherby et al ., 2009 ) have been implemented with success. Another major challenge is the decrease in diffraction lifetime, as radiation damage is more pronounced at non cryo-temperatures. Fortunately, data can be collected on multiple crystals, and the data merged into one single dataset to overcome this challenge ( Vaughan et al ., 2004 ). While shipping crystals is common for cryo-crystallography, most samples for room-temperature crystallography, including those grown for in-situ experiments, are grown at the X-ray source ( Axford et al ., 2012 , Bingel-Erlenmeyer et al ., 2011 , le Maire et al ., 2011 ). In fact, to our knowledge only one study exists detailing a means to ship room-temperature crystals, in this case for a membrane protein grown using in meso techniques ( Huang et al ., 2015 ). The Versatile Macromolecular Xtallography in-situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the highly automated collection of room-temperature X-ray diffraction data directly from 96-well crystallization plates, with the recommended plate type being MiTeGen In Situ-1™ ( Mikolajek et al ., 2023 , Sanchez-Weatherby et al ., 2019 ). The in-situ plate-based data collection method provides two major benefits for collecting room-temperature crystallographic data: it eliminates the need for physical manipulation of fragile crystals and reduces the risk of dehydration by maintaining a sealed environment. Prior to our experiments, there were three modes of access to users at VMXi: 1) users can travel with their pre-dispensed plates to the beamline, 2) users can travel to the crystallization facility which is part of the Research Complex at Harwell to set up plates, or 3) users can ship protein samples to the crystallization facility to be setup at DLS. Currently the majority of VMXi users are from within the United Kingdom, although there are a significant number of international users, which limits the accessibility of the beamline to worldwide researchers. While the shipping of crystals at 100 K in shipping dewars has become standardized and reliable, methods to achieve the same end without cryo-cooling have not yet been robustly established. This application provides a challenging test case for shipping samples in plates; not only must crystals arrive in good condition, excessive movement of the crystallization drop within the plates could cause crystals to move out of the field of view of the imaging system required for in situ data collection. This represents a more stringent test for shipping than simply shipping plates for crystal harvesting and mounting where movement of a drop, provided it remains intact, is less of a problem. Here, we present results showing the successful shipping of protein crystals, grown on MiTeGen In Situ-1 plates, from the University at Buffalo Hauptman Woodward Research Institute (UB-HWI) in Buffalo, New York, USA to DLS in Didcot, UK. We used 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 ( Figure 1 ). Our goal was to investigate whether shipping room-temperature crystals long distance, in this case internationally, using a standard shipping option from a commercial shipping company, has any impact on data quality. Identical crystallization plates were set up at UB-HWI and DLS with the well-characterized proteins lysozyme, thaumatin, and thermolysin, in order to compare the quality of diffraction and structures from crystals grown at each location. Our aim was to collect high-quality, room-temperature crystallographic data at VMXi for the crystals shipped that could be compared to data from samples set up on-site at DLS. Download figure Open in new tab Figure 1. Schematic overview of the experimental workflow. Samples were grown on MiTeGen In Situ-1 crystallization trays at UB-HWI, packed into the SSRL Blue Box thermal shipper, and transported by air and road approximately 3500 miles (5650 kilometers) via standard commercial shipping to DLS in the United Kingdom for room-temperature, in-situ X-ray diffraction experiments. We hypothesized that long-distance, room-temperature shipping of protein crystals might compromise data quality due to mechanical shock or thermal fluctuation. We found shipping the crystals transatlantic in the Blue Box resulted in no impact on data quality; we were able to solve comparable high-resolution structures from crystals grown at both locations. These results are promising in that they illustrate a way of transporting room-temperature crystal samples long-distance. These experiments provide proof-of-concept that not only simplifies room-temperature X-ray diffraction experiment set up, but also demonstrates a viable workflow for shipment of non-cryogenic protein crystals, which will allow more researchers from around the world to collect room-temperature crystallographic data at cutting edge beamlines. To our knowledge, this is the first comprehensive study on the effects of shipping at ambient temperature on protein crystals, particularly with the goal of in situ data collection, although other groups have shipped crystals at ambient temperature. 2. Methods 2.1. Crystallization Proteins were obtained as lyophilized powder and prepared for crystallization experiments as follows: Lysozyme (Hampton Research) was diluted to 35 mg/mL in 20 mM sodium acetate, pH 4.6. Thaumatin (Sigma Aldrich) was diluted to 50 mg/mL in distilled H2O. Thermolysin (Sigma Aldrich) was diluted to 40 mg/mL in 45% dimethylsulfoxide (DMSO), 20 mM Tris HCl pH 8.0. Initial crystallization conditions for thaumatin and thermolysin were determined by a high-throughput (HT) screen performed at the National Crystallization Center at UB-HWI ( Budziszewski et al ., 2023 , Lynch et al ., 2023 ). The HT conditions were determined using the micro-batch under oil method. Preliminary hits were optimized in 96-well ARI Intelli-plate vapor diffusion plates using a Formulatrix Formulator to generate cocktails and a Formulatrix NT8 to set up drops. Further optimization was performed for the MiTeGen In Situ-1 plates using the SPT Labtech Mosquito to dispense drops. Optimized crystallization conditions were as follows: Lysozyme was crystallized in 0.78 M sodium chloride and 0.1 M sodium acetate pH 4. Thaumatin was crystallized in 0.59 M di-ammonium tartrate. Thermolysin was crystallized in 0.20 M sodium di-hydrogen phosphate and 16.7% polyethylene glycol (PEG) 3350. Minimal optimization was required to translate crystallization conditions from the 96-well ARI Intelli-plate vapor diffusion plates to the MiTeGen In Situ-1 plates. For experimental setup at UB-HWI, optimized crystallization cocktails were dispensed into Greiner 96-well flat-bottom plates using the Formulatrix Formulator, with 35 microliters subsequently dispensed into MiTeGen In Situ-1 plates using a multi-channel pipette. Two 200 nL drops were set in each 96-well In Situ-1 plate using the SPT Labtech Mosquito at a 1:1 ratio of protein to cocktail. A single crystallization condition was used across all 96 wells for each protein sample. Plates were set up in duplicate for shipping. Additionally, Greiner 96-well flat-bottom plates with dispensed cocktails were shipped to Diamond Light Source along with lyophilized protein and buffer solution. For experimental setup at the Crystallisation Facility at Harwell Research Complex, lyophilized protein was prepared using the shipped buffers as stated above. Using a multi-channel pipette, 35 microliters of the optimized crystallization cocktails were dispensed from the shipped Greiner 96-well flat-bottom plate into MiTeGen In Situ-1 plates. Two 200 nL drops (1:1 protein to cocktail) were dispensed into each 96-well In Situ-1 plate using the SPT Labtech Mosquito. Care was taken to minimize procedural differences between plates set up at the two separate locations. 2.2. Shipping The SSRL Blue Box is optimized for shipping crystals or other temperature sensitive samples in microplates that conform to the SLAS standard footprint ( https://www.slas.org/education/ansi-slas-microplate-standards/ ). The SSRL beamline 12-1 user community has reliably used the Blue Box since 2020 to ship SSRL in-situ crystallization plates, compatible with an upgraded version of the Stanford Automated Mounter ( Russi et al ., 2016 ), for remote experiments with crystals maintained at controlled (non-cryogenic) temperature or controlled-humidity conditions. This includes successful commissioning shipments from Buffalo, NY to SSRL, Menlo Park, CA and subsequent diffraction data collection. The SSRL Blue Box consists of layers of impact isolating and thermally insulating materials ( Figure 2 ). It features an outer layer of corrugated blue plastic lined with flexible foam that surrounds a rigid box made from polyurethane insulation. Adjacent to each inside wall of the polyurethane box is a removable plastic liner that contains a non-toxic, phase-changing gel. Finally, the core of the Blue Box contains three stacks of foam holders with cut-outs that secure up to six standard microplates, each with an additional small gel pack on top, or six of the taller SSRL crystallization plates. Download figure Open in new tab Figure 2. In situ crystal transport set up. (A) Representative MiTeGen In Situ-1 plate dispensed with food coloring in the reservoir and dye for the crystallization drop (shown in grayscale for visualization). SSRL Blue Box external (B) and internal view with foam and insulation layers (C). Before shipping, the box—either with the lids open or just the removable phase-changing gel liners—should be conditioned at 21 ºC (±2 ºC) for at least 48 hours. Once packed, crystallization plates inside the Blue Box can maintain temperatures between 15 and 25 ºC for up to several (4-7) days, depending on ambient weather conditions. In a similar manner, a second temperature option, uses a different formulation of phase-changing gel to maintain temperatures between +2 and +8 °C. The optimized Blue Box assembly has been commercialized by the company Crystal Positioning Systems and is sold directly or by distributors. For our experiments, temperature packs were incubated at 23°C prior to packing samples. To remove potential difficulties from sample handling or shipment delays while in route, duplicate plates were packed into two separate SSRL Blue Box Thermal Shippers and sent over two consecutive days via commercial shipper. Samples cleared customs and arrived at DLS in 2-3 days from shipment date. 2.3. Data collection, processing, and structure determination Data were collected using the ISPyB system ( Delageniere et al ., 2011 ) employed by DLS ( Mikolajek et al ., 2023 ). Individual crystals were selected within the interface and queued for data collection. Collection parameters were 10 × 10 µm beam size, a flux of approximately 1e+12 ph/sec/mm 2 at 5% transmission, 0.0018s exposure, and a 60° rotation at 0.10° intervals for a total of 600 images with approximate X-ray diffraction weighted doses calculated using RADDOSE-3D ( Zeldin et al ., 2013 ) of ∼0.75 MGy. Datasets from multiple crystals auto-processed through the xia2.multiplex pipeline were selected for each experimental condition, and their xia2.multiplex statistics were averaged for comparison between crystallization set up location (US or UK). Scaled, unmerged mtz files from the xia2.multiplex analysis for representative datasets were downloaded from ISPyB and processed using the following programs within the CCP4i2 software suite ( https://www.ccp4.ac.uk ). Structures were solved using MolRep ( McCoy et al ., 2007 , Vagin & Teplyakov, 2010 ), and models rebuilt and refined using Coot ( Emsley & Cowtan, 2004 ) and Refmac5 ( Murshudov et al ., 2011 ). 3. Results 3.1. Setup and transportation of crystals For these experiments, entire 96-well plates were set up with a single protein and a single crystallization cocktail condition in order to generate a large number of similar crystals for each sample. Plates were imaged on the Formulatrix RockImager 1000 post-setup at UB-HWI for 4-5 days prior to shipment ( Figure 3A-C ). In an attempt to avoid shipping delays and mitigate potential rough handling of packages, duplicate crystallization plates were packaged in two separate SSRL Blue Boxes and shipped over two consecutive days to DLS in the United Kingdom. Plates arrived within 2-3 days of shipping. Download figure Open in new tab Figure 3. Images of crystals grown at UB-HWI in Buffalo, NY, imaged on UB-HWI’s Formulatrix Rock Imager 1000 (top row), imaged on DLS’s Formulatrix Rock Imager 1000 (middle row), and imaged on the VMXi beamline prior to data collection (bottom row). Upon arrival at DLS, plates were imaged using the Rock Imager 1000 at VMXi ( Figure 3D-F ). Representative images of crystals on which data were collected are shown in Figure 2 . Lysozyme and thaumatin crystals remained stationary in the plate during shipping, as seen in Figure 2 . Visual inspection revealed no notable differences between crystal droplets from the two shipment boxes. Thermolysin crystals shifted during shipping ( Figure 3F ), and further shifted when the plates were rotated from horizontal to vertical for placement on the beamline ( Figure 3I ). Interestingly, the drops remained in place even when crystals within those drops moved. The crystal movement can be attributed to the 22.5% DMSO that is present in the crystallization cocktail and importantly is a feature of plate reorientation for data collection rather than being related to the crystal shipping process itself. While the movement of the crystals in the drops did sometimes mean hitting an empty spot that had previously contained a crystal, we were able to collect data on thermolysin crystals that did not move out of the beam. It can also be seen that thaumatin and thermolysin crystals continued to grow during the shipping process ( Figure 3 ). Overall, no changes in crystal morphology or integrity were observed visually. Crystallization reservoir solution, lyophilized protein and protein buffers were shipped to DLS to replicate the protein setup at UB-HWI. Identical plates were set up at DLS’s Crystallisation Facility at Harwell in order to make comparisons on data quality. Overall, crystals grown at DLS were of similar visual quality to those grown at UB-HWI. 3.2. Room-temperature in-situ X-ray diffraction experiments at VMXi With the parameters used for data collection, minimal radiation damage was observed over the course of the experiment for all crystals. Each plate contained many crystals; therefore a few dozen datasets were collected for each experimental condition to ensure completeness from multicrystal datasets and to enable a robust comparison of data quality. VMXi recommends using the xia2.multiplex data when available, so we selected datasets processed through this pipeline for analysis ( Table 1 , Table S1). For lysozyme and thaumatin we compared 9 and 8 multi-crystal datasets respectively from UB-HWI and DLS grown crystals. Only 3 multi-crystal datasets were available for comparison for thermolysin due to the fact that crystals sank to the bottom of the well when the plate was rotated for placement on the beamline. While dozens of single crystal datasets were collected, auto processing only runs for multiple datasets collected from single crystallization drops. VMXi now supports grouping multiple drops for auto processing. It is important to note that while crystallization and data collection conditions were replicated as closely as possible, crystals grown at UB-HWI and shipped to DLS were slightly older in age prior to data collection. Overall, the data quality of the crystals are effectively the same, regardless of whether they were shipped from UB-HWI to DLS or set up in the UK. The resolution, CC1/2 values, and Rpim are broadly consistent, with minimal variability that is likely due to slight differences from the crystals on each plate in crystal size ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Comparison of average multiplex diffraction data quality. Lastly, we compared the space groups and unit cells of the crystals to ensure there were no alterations due to transportation conditions, for example if any dehydration had occurred. Crystals grown at both locations indexed into their expected space groups; lysozyme in P43212, thaumatin in P41212, and thermolysin in P6122. There were no major deviations measured in the unit cells calculated for crystals grown in either location (Table S1). These data indicate transatlantic shipping of RT protein crystals does not negatively affect the quality of diffraction data obtainable for the three crystal systems assessed. Crystals grown at UB-HWI and shipped long distance by air diffracted as well as those grown at DLS. From these datasets, a representative dataset was chosen for each protein and setup location to process further and ultimately deposit coordinates and data for into the PDB. These datasets generally had the best statistics from Table 1 , in terms of resolution and R-factor. Overall the data processing and statistics were very similar for crystals grown at each location for each of the 3 proteins (Tables S2-4). Notably, all structures were solved to approximately 2 Å or lower resolution. We also analyzed the B-factors throughout the main chain and saw little variation between structures. 4. Discussion Data collection from crystals maintained at cryogenic temperatures has dominated X-ray crystallography for the past three decades, generating a wealth of structural data to populate the Protein Data Bank, contributing to the development of significant advancements in structural biology, and providing the training data for the remarkable success of AlphaFold ( Jumper et al ., 2021 ). It offers numerous advantages including increased tolerance to radiation damage, as well as enabling efficient transport of samples from home labs to beamline facilities. However, cryogenic conditions can obscure biologically relevant conformations, in turn limiting insights into enzyme catalysis, ligand binding, and allostery. Recently, room-temperature crystallography is re-emerging as a desirable technique as it more closely represents physiological conditions. The major obstacles faced by room-temperature crystallography include the increased sensitivity to radiation damage, as well as the risk of crystal dehydration when crystals are removed from crystallization trays. These limitations highlight the need for improved experimental protocols. The VMXi beamline at DLS specializes in room-temperature crystallography in situ , i.e. measurements of diffraction data directly from crystals within crystallization plates. This technique is advantageous over other methods of room-temperature data collection because the crystals are never removed from their crystallization environment, and therefore are not subjected to stress from manipulation such as fracturing or dehydration. While we are not the first to ship crystals at ambient temperature, to our knowledge this study provides the first rigorous comparison of protein crystals grown and shipped at room temperature, transatlantic via air, with those set up and imaged locally at a synchrotron source. While cryogenic shipping of protein crystals is well-established and widely used, the development of reliable, non-cryogenic shipping protocols is essential for expanding the accessibility of room-temperature crystallography to the global community of structural biologists. Here, we show that high-resolution, room-temperature X-ray diffraction data can be collected from crystals shipped across the Atlantic Ocean. When comparing diffraction quality of crystals grown at UB-HWI and shipped to DLS to those grown at DLS, we observed no decrease in diffraction quality ( Table 1 ), and subsequently were able to determine high-quality structures of proteins from crystals grown at both facilities ( Figure 4 , Table S1). Download figure Open in new tab Figure 4. Representative structures with backbone alignment (top row), and representative view of side-chain electron density from 2Fo-Fc maps at 1.0 Sigma contour level for structures from crystals grown at UB-HWI (middle row) and at DLS (bottom row). Data quality statistics for structural models from crystals grown at UB-HWI and shipped to DLS are comparable to those for the sample setup at DLS. It was hypothesized that thermal and mechanical stresses of transport by air could result in a loss of diffraction quality, and changes to crystal integrity with regards to crystal packing and unit cell dimensions. Interestingly, virtually no changes in the unit cell of crystals grown at UB-HWI and transported to DLS compared to those grown at DLS were detected. We have previously noted variability reproducing crystals at different locations even with identical crystallization cocktail and protein prep. This is likely due to factors outside of our control, such as environmental humidity, or other facility-specific factors. Nonetheless, these factors did not negatively impact diffraction data quality. The ability to use crystals with minimal additional handling for X-ray diffraction experiments represents a major step forward towards unlocking structural information available in samples that are more difficult to study. Preparing crystals directly from initial optimization steps can enable a decrease in lab-to-lab variability and keeps sample preparation in the hands of the researcher all the way up to the step of measuring data. Importantly, these results illustrate an exciting new pipeline for growing and shipping crystals at room-temperature from the US to the VMXi beamline at DLS in the UK, and other cutting-edge beamlines around the world, increasing accessibility to room-temperature crystallography and empowering more studies on structural dynamics, fragment screening and ligand binding under noncryogenic conditions. Conflicts of interest The Thermal Shipper Blue Box is now available commercially from Crystal Positioning Systems ( www.crystalpositioningsystems.com ). Funding information Initial crystallization conditions for thaumatin and thermolysin crystals were determined with high-throughput screening at the National Crystallization Center at UB HWI (NIGMS R24GM141256). The research was supported by funding from the National Institute of General Medical Sciences of the National Institutes of Health (R01GM141273). The Bowman Lab is a Member of the SBGrid Consortium ( Morin et al ., 2013 ). The Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). Development of the Blue Box was also supported through the DOE-STTR program. The crystallisation facility at Harwell is supported by Diamond Light Source Ltd, The Rosalind Franklin Institute and The Medical Research Council. This work was carried out with the support of Diamond Light Source instrument VMXi (proposal nt39424). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. Funder Information Declared National Institute of General Medical Sciences, https://ror.org/04q48ey07 , R01GM141273 , P30GM133894 , R24GM141256 Office of Basic Energy Sciences, https://ror.org/05mg91w61 , DE-AC02-76SF00515 Diamond Light Source, https://ror.org/05etxs293 , nt39424S Rosalind Franklin Institute, https://ror.org/01djcs087 Medical Research Council Office of Biological and Environmental Research, https://ror.org/0114b2m14 United States Department of Energy, https://ror.org/01bj3aw27 Footnotes Synopsis The effect of international shipment of protein crystals on room-temperature X-ray diffraction quality. References ↵ Axford , D. , Owen , R. L. , Aishima , J. , Foadi , J. , Morgan , A. W. , Robinson , J. I. , Nettleship , J. E. , Owens , R. J. , Moraes , I. , Fry , E. E. , Grimes , J. M. , Harlos , K. , Kotecha , A. , Ren , J. , Sutton , G. , Walter , T. S. , Stuart , D. I. & Evans , G. ( 2012 ). In situ macromolecular crystallography using microbeams . 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Journal of Applied Crystallography 23 , 215 – 218 . OpenUrl CrossRef Web of Science ↵ Zeldin , O. B. , Gerstel , M. & Garman , E. F. ( 2013 ). Raddose-3d: Time- and space-resolved modelling of dose in macromolecular crystallography . Journal of Applied Crystallography 46 , 1225 – 1230 . OpenUrl CrossRef Web of Science View the discussion thread. Back to top Previous Next Posted June 17, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Does crossing the pond affect crystal quality? Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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