The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

Mathews, I.I.; Allison, Kim; Robbins, Thomas; Lyubimov, A.Y.; Uervirojnangkoorn, Monarin; Brunger, Axel T.; Khosla, Chaitan; DeMi̇rci̇, Hasan; McPhillips, S.E.; Hollenbeck, Michael; Soltis, S. Michael; Cohen, Aina E.

doi:10.1021/acs.biochem.7b00711

Cited by 22 publications

(17 citation statements)

References 20 publications

(34 reference statements)

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“…Even where major conformational differences are absent, circumventing non-isomorphism problems lowers errors in structure-factor measurements, and clustering data sets could provide a valuable means for identifying systematic errors (Diederichs, 2017). The general importance of redundancy in data collection is increasingly being recognized, and serial crystallography experiments are becoming routine Weinert et al, 2017;Mathews et al, 2017;Standfuss & Spence, 2017), but averaging measurements of potentially nonequivalent physical entities (for example structure factors from different regions of a crystal) may be detrimental. The improvement in data quality that can be gained by clustering multiple related data sets into isomorphous groups during data reduction has been explored previously in several contexts (Liu et al, 2011;Giordano et al, 2012;Foadi et al, 2013;Zander et al, 2016;Assmann et al, 2016;Diederichs, 2017;Yamamoto et al, 2017), although previous work has generally focused on non-isomorphism resulting from changes to crystal-packing interactions, rather than emphasizing the potential to use this feature of protein crystals as a means to explore conformational heterogeneity.…”

Section: Discussionmentioning

confidence: 99%

Microfocus diffraction from different regions of a protein crystal: structural variations and unit-cell polymorphism

Thompson

Cascio²,

Yeates

2018

Acta Crystallogr D Struct Biol

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Real macromolecular crystals can be non-ideal in a myriad of ways. This often creates challenges for structure determination, while also offering opportunities for greater insight into the crystalline state and the dynamic behavior of macromolecules. To evaluate whether different parts of a single crystal of a dynamic protein, EutL, might be informative about crystal and protein polymorphism, a microfocus X-ray synchrotron beam was used to collect a series of 18 separate data sets from non-overlapping regions of the same crystal specimen. A principal component analysis (PCA) approach was employed to compare the structure factors and unit cells across the data sets, and it was found that the 18 data sets separated into two distinct groups, with large R values (in the 40% range) and significant unit-cell variations between the members of the two groups. This categorization mapped the different data-set types to distinct regions of the crystal specimen. Atomic models of EutL were then refined against two different data sets obtained by separately merging data from the two distinct groups. A comparison of the two resulting models revealed minor but discernable differences in certain segments of the protein structure, and regions of higher deviation were found to correlate with regions where larger dynamic motions were predicted to occur by normal-mode molecular-dynamics simulations. The findings emphasize that large spatially dependent variations may be present across individual macromolecular crystals. This information can be uncovered by simultaneous analysis of multiple partial data sets and can be exploited to reveal new insights about protein dynamics, while also improving the accuracy of the structure-factor data ultimately obtained in X-ray diffraction experiments.

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Section: Discussionmentioning

confidence: 99%

Microfocus diffraction from different regions of a protein crystal: structural variations and unit-cell polymorphism

Thompson

Cascio²,

Yeates

2018

Acta Crystallogr D Struct Biol

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“…Other implementations have also demonstrated their proof-of-concepts as viable sample delivery methods for serial crystallography. In the crystal extractor [166], crystals were extracted onto a mesh or thin-film substrate and carried into the path of the X-ray beam. Diffraction data is collected by raster scanning of the substrate, followed by stringent data processing criteria to avoid including diffraction images from crystals contaminated from a previous exposure.…”

Section: Other Sample Delivery Methodsmentioning

confidence: 99%

“…This process is repeated by dipping the substrate back into the crystal suspension until no more diffraction data could be recorded, indicating that the crystal supply has been exhausted. As crystals are sealed in a container in their native mother liquor solution, the crystal extractor has been shown to be suitable for polyketides synthases crystals, which are sensitive to mechanical stress and enabled high resolution structural data to be collected at room temperature [166]. As crystal diffraction volume decreases the detrimental effect of background contribution increases.…”

Section: Other Sample Delivery Methodsmentioning

confidence: 99%

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Cheng¹

2020

Crystals

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The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.

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“…An important capability of the XFEL experiment is the ability to use very small crystals at room temperature (RT) to produce useful data sets beyond what is currently possible at the synchrotron. This makes SFX ideally suited to study samples that elude crystal growth to larger size (Mathews et al, 2017;Boudes et al, 2016) or to study water networks and alternative side-chain conformations within protein crystals that are disrupted by cryopreservation (Thomaston et al, 2017;Keedy et al, 2015). Crystallization approaches thus move from generating one perfect crystal for a traditional rotation experiment under cryoconditions to batch crystallization approaches for the preparation of large amounts of good-quality microcrystals to nanocrystals.…”

Section: Crystal-size Considerationsmentioning

confidence: 99%

“…The crystal extractor has been developed to deliver crystals directly from mother-liquor solutions at ambient temperature and pressure, and has been demonstrated at the XFEL and the synchrotron (Mathews et al, 2017). The method is compatible with a mixture of crystal sizes and is simple to set up and operate.…”

Section: Crystal Extractormentioning

confidence: 99%

Strategies for sample delivery for femtosecond crystallography

Martiel

Müller‐Werkmeister

Cohen

2019

Acta Crystallogr D Struct Biol

Self Cite

103

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Highly efficient data-collection methods are required for successful macromolecular crystallography (MX) experiments at X-ray free-electron lasers (XFELs). XFEL beamtime is scarce, and the high peak brightness of each XFEL pulse destroys the exposed crystal volume. It is therefore necessary to combine diffraction images from a large number of crystals (hundreds to hundreds of thousands) to obtain a final data set, bringing about sample-refreshment challenges that have previously been unknown to the MX synchrotron community. In view of this experimental complexity, a number of sample delivery methods have emerged, each with specific requirements, drawbacks and advantages. To provide useful selection criteria for future experiments, this review summarizes the currently available sample delivery methods, emphasising the basic principles and the specific sample requirements. Two main approaches to sample delivery are first covered: (i) injector methods with liquid or viscous media and (ii) fixed-target methods using large crystals or using microcrystals inside multi-crystal holders or chips. Additionally, hybrid methods such as acoustic droplet ejection and crystal extraction are covered, which combine the advantages of both fixed-target and injector approaches.

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The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

Cited by 22 publications

References 20 publications

Microfocus diffraction from different regions of a protein crystal: structural variations and unit-cell polymorphism

Microfocus diffraction from different regions of a protein crystal: structural variations and unit-cell polymorphism

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Strategies for sample delivery for femtosecond crystallography

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