Development of high-performance X-ray transparent crystallization plates for<i>in situ</i>protein crystal screening and analysis

Soliman, Ahmed; Warkentin, Matthew; Apker, Benjamin; Thorne, Robert

doi:10.1107/s090744491101883x

Cited by 25 publications

(22 citation statements)

References 34 publications

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“…In particular, COC has seen widespread adoption as the polymer film of choice for X-ray compatible devices, 46 including simple channel structures for counterdiffusion, 56,58,60,61,126 droplet-based devices, 108,109,118 and larger-scale X-ray compatible wellplates. [127][128][129][130][131][132][133][134][135][136][137][138][139][140] However, further decreasing the device thickness to achieve the signal-to-noise levels required for microcrystallography is a significant materials' challenge. Typical reports of X-ray compatible microfluidics describe results where the path length of the device materials is nearly twice that of the crystal of interest.…”

Section: B Device Materials For Microcrystallographymentioning

confidence: 99%

Microfluidics: From crystallization to serial time-resolved crystallography

Sui

Perry

2017

Structural Dynamics

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Capturing protein structural dynamics in real-time has tremendous potential in elucidating biological functions and providing information for structure-based drug design. While time-resolved structure determination has long been considered inaccessible for a vast majority of protein targets, serial methods for crystallography have remarkable potential in facilitating such analyses. Here, we review the impact of microfluidic technologies on protein crystal growth and X-ray diffraction analysis. In particular, we focus on applications of microfluidics for use in serial crystallography experiments for the time-resolved determination of protein structural dynamics.

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Section: B Device Materials For Microcrystallographymentioning

confidence: 99%

Microfluidics: From crystallization to serial time-resolved crystallography

Sui

Perry

2017

Structural Dynamics

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“…In most laboratories this harvesting step remains the same delicate, labour-intensive, manual process that it was at the advent of cryocrystallograpy (Garman & Schneider, 1997). One strategy to eliminate the mounting bottleneck has been to avoid the need for transfer entirely, by developing in situ diffraction techniques (Bingel-Erlenmeyer et al, 2011;Michalska et al, 2015;Soliman, Warkentin, Apker, & Thorne, 2011). Other approaches to ex situ screening have tried to design human out of the process, via novel harvesting techniques, or by reproducing the human mounting technique with advanced robotics (Cipriani et al, 2012;Deller & Rupp, 2014;Viola et al, 2011;Viola, Carman, Walsh, Frankel, & Rupp, 2007).…”

Section: Introductionmentioning

confidence: 99%

The Low-Cost, Semi-Automated Shifter Microscope Stage Transforms Speed and Robustness of Manual Protein Crystal Harvesting

Wright

Collins

Talon

et al. 2019

Preprint

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Synopsis A motorised X/Y microscope stage is presented that combines human fine motor control with machine automation and automated experiment documentation, to transform productivity in protein crystal harvesting. AbstractDespite the tremendous success of x-ray cryocrystallography over recent decades, the transfer of crystals from the drops where they grow to diffractometer sample mounts, remains a manual process in almost all laboratories. Here we describe the Shifter, a semi-automated microscope stage that offers an accessible and scalable approach to crystal mounting that exploits on the strengths of both humans and machines. The Shifter control software manoeuvres sample drops beneath a hole in a clear protective cover, for human mounting under a microscope. By allowing complete removal of film seals the tedium of cutting or removing the seal is eliminated. The control software also automatically captures experimental annotations for uploading to the user's data repository, removing the overhead of manual documentation. The Shifter facilitates mounting rates of 100-240 crystals per hour, in a more controlled process than manual mounting, which greatly extends the lifetime of drops and thus allows for a dramatic increase in the number of crystals retrievable from any given drop, without loss of X-ray diffraction quality. In 2015 the first in a series of three Shifter devices was deployed as part of the XChem fragment screening facility at Diamond Light Source (DLS), where Acta Crystallographica Section D research papers 2 they have since facilitated the mounting of over 100,000 crystals. The Shifter was engineered to be simple, allowing for a low-cost device to be commercialised and thus potentially transformative as many research initiatives as possible.

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“…Therefore, novel approaches are being sought by engineering the sample environment such that diffraction data of crystals can be collected within their growth environment. One such method is termed in situ or plate-screening 23,24 and it is already implemented at a number of macromolecular crystallography beamlines at various synchrotron sources worldwide 25 . However, the use of this method is limited by the geometrical parameters of the crystal plate and the space available around the sample point of the instrument.…”

Section: Introductionmentioning

confidence: 99%

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Feiler

Wallacher

Weiss

2019

JoVE

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Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

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Development of high-performance X-ray transparent crystallization plates forin situprotein crystal screening and analysis

Cited by 25 publications

References 34 publications

Microfluidics: From crystallization to serial time-resolved crystallography

Microfluidics: From crystallization to serial time-resolved crystallography

The Low-Cost, Semi-Automated Shifter Microscope Stage Transforms Speed and Robustness of Manual Protein Crystal Harvesting

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

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