Best practices for time-resolved serial synchrotron crystallography

Schulz, Eike C.; Yorke, Briony A.; Pearson, Arwen R.; Mehrabi, P.

doi:10.1107/s2059798321011621

Cited by 37 publications

(17 citation statements)

References 123 publications

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“…Here, the relative error increases with decreasing time-delays and in consequence the comparability between different samples is reduced. While true time-resolved crystallographic experiments at room temperature offer a much more complete access to the dynamic landscape of protein function, cryo-trapping experiments may be sufficient to solve many important biologically relevant questions—e.g., provide insight into thermodynamically trapped, stable reaction intermediates 22 , 23 . It is clear that care has to be taken to only use crystals of highly similar dimensions to avoid that mechanistic conclusions are affected by different diffusion times.…”

Section: Discussionmentioning

confidence: 99%

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

et al. 2023

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We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.

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Section: Discussionmentioning

confidence: 99%

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

et al. 2023

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“…In time-resolved experiments, protein molecules inside the crystal need to be triggered, typically by a pump laser but potentially also a substrate solution (Schulz et al, 2022). The size and number of crystals in a fixed-target cavity will greatly affect the activation efficiency of a trigger as they will determine its penetration into the crystals.…”

Section: A Question Of Crystal Sizementioning

confidence: 99%

Micro-structured polymer fixed targets for serial crystallography at synchrotrons and XFELs

Carrillo,

Mason,

Karpik

et al. 2023

IUCrJ

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Fixed targets are a popular form of sample-delivery system used in serial crystallography at synchrotron and X-ray free-electron laser sources. They offer a wide range of sample-preparation options and are generally easy to use. The supports are typically made from silicon, quartz or polymer. Of these, currently, only silicon offers the ability to perform an aperture-aligned data collection where crystals are loaded into cavities in precise locations and sequentially rastered through, in step with the X-ray pulses. The polymer-based fixed targets have lacked the precision fabrication to enable this data-collection strategy and have been limited to directed-raster scans with crystals randomly distributed across the polymer surface. Here, the fabrication and first results from a new polymer-based fixed target, the micro-structured polymer fixed targets (MISP chips), are presented. MISP chips, like those made from silicon, have a precise array of cavities and fiducial markers. They consist of a structured polymer membrane and a stabilization frame. Crystals can be loaded into the cavities and the excess crystallization solution removed through apertures at their base. The fiducial markers allow for a rapid calculation of the aperture locations. The chips have a low X-ray background and, since they are optically transparent, also allow for an a priori analysis of crystal locations. This location mapping could, ultimately, optimize hit rates towards 100%. A black version of the MISP chip was produced to reduce light contamination for optical-pump/X-ray probe experiments. A study of the loading properties of the chips reveals that these types of fixed targets are best optimized for crystals of the order of 25 µm, but quality data can be collected from crystals as small as 5 µm. With the development of these chips, it has been proved that polymer-based fixed targets can be made with the precision required for aperture-alignment-based data-collection strategies. Further work can now be directed towards more cost-effective mass fabrication to make their use more sustainable for serial crystallography facilities and users.

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“…As a demonstration of the importance of these tools, and their future development, they were instrumental in facilitating the rapid solution and availability of numerous protein-ligand structures of several proteins from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the wake of the coronavirus disease (COVID-19) pandemic ( Douangamath et al, 2020 ; Günther et al, 2021 ; Newman et al, 2021 ; Schuller et al, 2021 ; Kozielski et al, 2022 ). However, availability of these programs is currently restricted to certain synchrotron sites and they do not allow for customized workflow configuration or provide interfaces for definition of new experiments ( Moreno-Chicano et al, 2019 ; Brändén and Neutze, 2021 ; Schulz et al, 2022 ).…”

Section: Batch Data Processing and Refinement Toolsmentioning

confidence: 99%

Experiences From Developing Software for Large X-Ray Crystallography-Driven Protein-Ligand Studies

Pearce

Skyner²,

Krojer

2022

Front. Mol. Biosci.

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The throughput of macromolecular X-ray crystallography experiments has surged over the last decade. This remarkable gain in efficiency has been facilitated by increases in the availability of high-intensity X-ray beams, (ultra)fast detectors and high degrees of automation. These developments have in turn spurred the development of several dedicated centers for crystal-based fragment screening which enable the preparation and collection of hundreds of single-crystal diffraction datasets per day. Crystal structures of target proteins in complex with small-molecule ligands are of immense importance for structure-based drug design (SBDD) and their rapid turnover is a prerequisite for accelerated development cycles. While the experimental part of the process is well defined and has by now been established at several synchrotron sites, it is noticeable that software and algorithmic aspects have received far less attention, as well as the implications of new methodologies on established paradigms for structure determination, analysis, and visualization. We will review three key areas of development of large-scale protein-ligand studies. First, we will look into new software developments for batch data processing, followed by a discussion of the methodological changes in the analysis, modeling, refinement and deposition of structures for SBDD, and the changes in mindset that these new methods require, both on the side of depositors and users of macromolecular models. Finally, we will highlight key new developments for the presentation and analysis of the collections of structures that these experiments produce, and provide an outlook for future developments.

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Best practices for time-resolved serial synchrotron crystallography

Cited by 37 publications

References 123 publications

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Micro-structured polymer fixed targets for serial crystallography at synchrotrons and XFELs

Experiences From Developing Software for Large X-Ray Crystallography-Driven Protein-Ligand Studies

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