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

Abstract

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.

Fragment Binding to the Nsp3 Macrodomain of SARS-CoV-2 Identified Through Crystallographic Screening and Computational Docking

“…Despite having generally weak overall binding affinity, fragments often have high ligand efficiency, and can provide templates for further chemical elaboration into lead-like molecules (29,30). Crystallography can be used as a primary screening method for fragment discovery (31), and recent automation and processing software at synchrotron radiation sources has made this routinely possible at facilities like the XChem platform at Diamond Light Source (32)(33)(34)(35)(36)(37). As part of Diamond's contribution toward efforts to combat COVID-19, fragment screening expertise and infrastructure was made immediately available to any users working on SARS-CoV-2 targets (38).…”

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

“…While the screening experiment itself has long relied on the high throughput of solution methods like NMR or SPR 23 , rapid advances in technology and automation at synchrotron radiation sources 24 has made screening directly in crystal structures routinely possible at facilities like the XChem platform at Diamond Light Source [25][26][27][28] . These have been further enhanced by techniques such as mass spectrometry for the discovery of covalently binding fragments 29 .…”

Changes in Membrane Protein Structural Biology

“…The mounting of an MP crystal in a loop is still a mainly manual exercise, although there have been developments towards automated mounting. These include the fully automated Crystaldirect approach which relies on using cyclic olefin copolymer (COC) film plates and excising whole wells and the semi-automated shifter system that enables individual crystals to be mounted at 6 x the rate of a standard mounting session [201,202]. COC film plates have also been developed for LCP crystallisation of MPs [203][204][205].…”