Hitting the target: fragment screening with acoustic<i>in situ</i>co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

Yin, Xingyu; Scalia, A.; Leroy, Ludmila; Cuttitta, Christina M.; Polizzo, G.M.; Ericson, Daniel; Roessler, Christian G.; Campos, O.; Millie, Y.; Agarwal, Rashmi; Jackimowicz, R.; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

doi:10.1107/s1399004713034603

Cited by 49 publications

(52 citation statements)

References 54 publications

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“…On micro-meshes, as many as 10 protein crystals can be combined with 10 different fragments [6]. An unlimited number of crystal + fragment screens can be combined on a conveyor belt [5].…”

Section: Discussionmentioning

confidence: 99%

A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

et al. 2014

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High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.

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“…On micro-meshes, as many as 10 protein crystals can be combined with 10 different fragments [6]. An unlimited number of crystal + fragment screens can be combined on a conveyor belt [5].…”

Section: Discussionmentioning

confidence: 99%

A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

et al. 2014

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“…In a related note, a recent publication reported an elaboration of this idea to enable crystallography-based screening directly using ADE to position multiple crystallization drops in situ on x-ray source micromesh mounts. 8 Although this required some customized hardware, it bypasses manual steps of crystal mounting and streamlines crystal exchange time during synchrotron data collection. The efficacy of these approaches is dependent on a relatively robust crystallization system.…”

Section: Discussionmentioning

confidence: 99%

“…We further describe parameters of an integrated protein crystallization system that includes the Echo ADE supported by a robotic arm, plate stackers, plate peeler, plate sealer, and centrifuge. Acoustic technology has potential impact in other stages of protein crystallography, documented in prior and ongoing creative examples of microcrystal suspension transfer to aid seeding of protein crystallization, 6 mounting crystals for data collection, 7 growing crystals in situ for direct data collection to enable efficient structure-based screening of ligands, 8 and delivering nanocrystals for x-ray free electron laser analyses.…”

Section: This Issue) As Implemented In the Labcytementioning

confidence: 99%

Developments in the Implementation of Acoustic Droplet Ejection for Protein Crystallography

Ping¹,

Noland²,

Ultsch³

et al. 2016

SLAS Technology

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Acoustic droplet ejection (ADE) enables crystallization experiments at the low-nanoliter scale, resulting in rapid vapor diffusion equilibration dynamics and efficient reagent usage in the empirical discovery of structure-enabling protein crystallization conditions. We extend our validation of this technology applied to the diverse physicochemical property space of aqueous crystallization reagents where dynamic fluid analysis coupled to ADE aids in accurate and precise dispensations. Addition of crystallization seed stocks, chemical additives, or small-molecule ligands effectively modulates crystallization, and we here provide examples in optimization of crystal morphology and diffraction quality by the acoustic delivery of ultra-small volumes of these cofactors. Additional applications are discussed, including set up of in situ proteolysis and alternate geometries of crystallization that leverage the small scale afforded by acoustic delivery. Finally, we describe parameters of a system of automation in which the acoustic liquid handler is integrated with a robotic arm, plate centrifuge, peeler, sealer, and stacks, which allows unattended high-throughput crystallization experimentation.

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“…Faced with the inevitable trade-offs between different capabilities, we were motivated to begin finding answers to some of these questions. Our group has successfully demonstrated unique new approaches to serial micro-crystallography, such as acoustic droplet ejection (Ellson et al, 2003), which uses sound waves to move crystals directly from their growth trays onto data-collection media (Soares et al, 2011;Roessler et al, 2013) or to grow protein crystals on plates (Villaseñ or et al, 2012) or directly on micro-meshes (Yin et al, 2014). Initially, our serial crystallography efforts were frustrated by difficulties such as preferential orientation and crystal clustering.…”

Section: Introductionmentioning

confidence: 99%

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes,in situplates and on a movable crystal conveyor belt

et al. 2014

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X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes 2 , Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6 wedges of data were taken from $100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the datacollection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 mm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. Highquality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.

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Hitting the target: fragment screening with acousticin situco-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

Cited by 49 publications

References 54 publications

A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

Developments in the Implementation of Acoustic Droplet Ejection for Protein Crystallography

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes,in situplates and on a movable crystal conveyor belt

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