Biology subjects

Sanchez-Weatherby, J.

Publications and source records attributed to Sanchez-Weatherby, J..

3 recordsLinked to original sources

bioRxiv2025

Hundred-Picolitre Droplet on Demand Tape Drive and XES Prototypes for Time-Resolved Serial Crystallography at VMXi, Diamond Light Source

Time resolved X-ray crystallography is experiencing a resurgence, in part, because of serial methods that readily allow scientists to create stop-motion movies of macromolecular function of photoactivation, enzyme catalysed reactions, and ligand-induced conformational changes triggering further downstream signalling events. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet on demand tape drive sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with electronic states of metal ions within the sample. To our knowledge, such a combined methodology has not been deployed previously at a synchrotron beamline and has been restricted to XFELs. Here we describe prototype experiments along the development pathway to a combined droplet on demand diffraction and XES system at the microfocus beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within hundreds of picolitre-volume droplets deposited on a moving tape. In separate experiments at VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high viscosity extruder. Together, these results demonstrate the feasibility of combined droplet on demand serial crystallography and XES experiments using a third-generation synchrotron beamline; project work currently underway at Diamond Light Source. SynopsisWe describe proof of concept experiments towards correlated serial crystallography (SSX) and X-ray emission spectroscopy (XES) from microcrystals at a microfocus synchrotron beamline. A droplet on demand tape drive system delivers microcrystals to the beam within well-separated, hundreds of picolitre-volume droplets while XES allows validation of redox states of metals within protein crystals.

biophysics↗

bioRxiv2025

Does crossing the pond affect crystal quality?

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.

biophysics↗

bioRxiv2022

xia2.multiplex: a multi-crystal data analysis pipeline

In macromolecular crystallography radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals, for example small-wedge data collections on micro-crystals, in situ room-temperature data collections, and collection from membrane proteins in lipidic mesophase. Whilst indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. Identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help optimise the scaling and merging of multiple data sets, we developed a new program, xia2.multiplex, which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multicrystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After describing a number of use cases, we demonstrate the benefit of xia2.multiplex within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment screening experiments on the SARS-CoV-2 main protease.

molecular biology↗