Biology subjects

Bosman, R.

Publications and source records attributed to Bosman, R..

3 recordsLinked to original sources

bioRxiv2026

Best practices for cryo-trapping time-resolvedcrystallography with the Spitrobot crystal plunger

Capturing meta-stable conformations of enzymes and ligand complexes demands structural snapshots beyond static crystal structures. While time-resolved serial crystallography at room temperature, offers a time-resolution down to the femto-second domain it requires large amounts of micro crystals, specialized beamlines and considerable experience. Moreover, as the majority of enzymes displays turnover-times in the millisecond domain or slower, simpler methods can provide meaningful structural insight into enzyme catalysis. Vitrification of protein crystals can trap reaction intermediates by rapid cooling to {inverted exclamation} 100 K, and has traditionally been used to gain insight into long lived reaction intermediates such as product complexes. However, manual vitrification procedures are limited to long delay times of at least several seconds and heavily suffer from operator variability. A solution to this problem is provided by automatic crystal plunging devices, such as the Spitrobot, that plunge loop-mounted protein crystals into liquid nitrogen within millisecond time-scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light-sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo-trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high-throughput capabilities. Thus, compared to room-temperature time-resolved crystallography, users not only benefit from uncoupling sample-preparation and data-collection, but also from a reduction in the required technical expertise and ready access to radiation sources. However, as cryo-trapping crystallography explores dynamic structural changes that become only visible by the comparison of several samples, experiments have to be carefully planned to carry out the necessary controls and to avoid mis- or over-interpretation of the results. Here we describe a detailed protocol for cryo-trapping time-resolved crystallography using automated crystal-plungers that enables researchers to map enzymatic reaction coordinate pathways within the millisecond domain.

biochemistry↗

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↗

bioRxiv2019

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The structures reveal a twist of the D-ring, which lead to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signalling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the water dissociation is key to the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

biophysics↗