Chapter 1. Practical Approaches for <i>In Situ</i> X-ray Crystallography: from High-throughput Screening to Serial Data Collection

Martiel, Isabelle; Oliéric, V.; Caffrey, Martin; Wang, Meitian

doi:10.1039/9781788010504-00001

Cited by 3 publications

(2 citation statements)

References 76 publications

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“…Although the traditional LCP sandwich glass plate enables high throughput screening of crystallization conditions, the glass sandwich plate cannot be used directly for X-ray diffraction screening due to poor X-ray transparency and strong scattering. Therefore, other materials have been explored to lower X-ray background scattering and increase transmission (43). Thin plastic polymers have been tested including polydimethylsiloxane (PDMS, 14-45 µm), polyvinyl chloride (PVC, 50 µm), polyethylene terephthalate (PET, 50 µm) and cyclic olefin copolymer (COC, 50-100 µm), and applications have been reported in microfluidic devices for on-chip characterization (44)(45)(46) and for the data collection on MPs (47,48).…”

Section: Imisx Platementioning

confidence: 99%

In Meso In Situ Serial X-Ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source

Huang

Oliéric

Caffrey

et al. 2020

Methods in Molecular Biology

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The lipid cubic phases (LCP) has enabled the determination of many important high-resolution structures of membrane proteins such as G protein-coupled receptors, photosensitive proteins, enzymes, channels and transporters. However, harvesting the crystals from the glass or plastic plates in which crystals grow is challenging. The in meso in situ serial X-ray crystallography (IMISX) method uses thin plastic windowed plates that minimize LCP crystal manipulation.The method, which is compatible with high-throughput in situ measurements, allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting. In this chapter, we describe a IMISX protocol for in situ serial X-ray data collection of LCP-grown crystals at both cryogenic and room temperatures which includes the crystallization setup, sample delivery, automated serial diffraction data collection and experimental phasing. We also detail how the IMISX method was applied successfully for the structure determination of two novel targets -the undecaprenyl-pyrophosphate phosphatase BacA and the chemokine G protein-coupled receptor CCR2A.

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Section: Imisx Platementioning

confidence: 99%

In Meso In Situ Serial X-Ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source

Huang

Oliéric

Caffrey

et al. 2020

Methods in Molecular Biology

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“…The original design and implementation of the beamline have been previously described (Sanchez-Weatherby et al, 2019) together with examples of initial data measured from thaumatin crystals in situ within crystallization plates. Subsequently, other beamlines have been developed with similar features (Okumura et al, 2022;Martiel et al, 2018) in parallel with microfluidic and serial crystallographic approaches (Martiel et al, 2019(Martiel et al, , 2018. Here, we describe the current status of the protein crystallization facility (PXF) and VMXi, and provide representative use cases, indicating the benefits of a highly automated RT datacollection pipeline.…”

Section: Introductionmentioning

confidence: 98%

Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi

Mikolajek¹,

Sánchez-Weatherby²,

Sandy³

et al. 2023

Int Union Crystallogr J

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The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.

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In situ crystal data-collection and ligand-screening system at SPring-8

Okumura¹,

Sakai²,

Murakami³

et al. 2022

Acta Cryst Sect F

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In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein–ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser.

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Chapter 1. Practical Approaches for In Situ X-ray Crystallography: from High-throughput Screening to Serial Data Collection

Cited by 3 publications

References 76 publications

In Meso In Situ Serial X-Ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source

In Meso In Situ Serial X-Ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source

Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi

In situ crystal data-collection and ligand-screening system at SPring-8

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