Nathan D. Wrighta, Patrick Collinsb, Romain Talona and Frank von Delftba*
aStructural Genomics Consortium, University of Oxford, ORCRB, Roosevelt Drive, Oxford, OX3 7DQ, UK
b Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, OX11 0QX, United Kingdom
Correspondence email: firstname.lastname@example.org
The majority of protein crystals, used for structural determination by x-ray diffraction, are grown in a drop, in a crystallisation plate, and are removed from the drop to a sample mount in a process of manual harvesting by skilled human ‘Mounters’. Whether it is carried out on a small scale by individual scientist in broad-interest group, or by teams of crystallographers in a high throughput facility, manual harvesting represents a bottleneck in the crystallography workflow. Scientists are faced with the challenges of data management, of experimental variability as the crystal is removed from the drop, and of drop deterioration when extracting multiple crystals for a single drop.
A novel, automated X/Y stage, with capacity for two SBS plates, called the Shifter, has been developed to address this short-coming. The main enclosure of the device (approx. 90cm x 30cm x 6cm) is installed at a suitable harvesting microscope, between the microscope base, and the objective lens.
The lid or ‘Shroud’ of the enclosure is of clear acrylic, with a port for loading plates, and an 8mm working aperture, concentric with the optical axis of the microscope, for access to the crystal drop.
Whilst all experimentalists will be concerned with experimental variability, as the need for crystal harvesting is scaled up to the high throughput setting, current manual harvesting practices become rate limiting. In Autumn 2015 the Shifter was been deployed to the XChem fragment screening facility (MX Beamline I04-1, Diamond Light Source (DLS)), to facilitate a collaboration between SGC Oxford(PX) and I04-1 (DLS) to routinely to screen libraries of weakly-binding compounds (‘fragments’). The number of crystals harvested per target is proportionally higher for ‘Fragment Based Drug Discovery’ campaigns (excess of 600 per target), than conventional crystallisation trials (28 per structure across the SGC), and usually involved additional ‘interventions’ such as fragment or cryoprotectant dispensing.
· Harvesting Mode: Pick lists generated in .CSV format, from any upstream database, are loaded into the system GUI. Autocompletion of tracking information has eliminated administrative tasks so that taken to fill a 16 pin Uni-Puck has dropped from 60-80 minutes, to 10 minutes.
· Form buttons allow routine capturing of failure/success modes for crystal characterisation, via a touch-screen monitor.
· Soak Mode: Parallel lists of crystal locations and soak locations are loaded. As the user navigates between the two lists blank columns record associated crystal identities, soak conditions and duration; these are exported as a .csv file, for loading into a database.
Since being deployed at XChem >45,000 samples have been mounted. Harvesting rates of 100 pX/hour are now considered routine, with rates of >240 pX/hour achievable for complaisant systems in short bursts.
1. Krojer, T., Talon, R., Collins, P., Cox, O., Fairhead, M., Strain-Damerell, C., Bradley, A., Pearce, N, Ng, J.T., Wright, N.D., Sethi, R., Szykowska, A., Wiggers, H., Douangamath, A., Brandao-Neto, J., Bowkett, D, Burgess-Brown, N., Marsden, B., Brennan, P., von Delft, F. (2015) Routine Fragment Screening by Crystal Structure. Unpublished poster presentation at: Fragments 2015: Fifth RSC-BMCS Fragment-based Drug Discovery meeting. 22 - 24 March 2015, Cambridge, United Kingdom
2. McEwan, P.A., Collins, P., Dias, A., Mazanetz, M., Brandao-Neto, B., Douangamath, A., Ng, J.T., Wright, N.D., Krojer, T, Talon R., Barker, J, von Delft, F. (2015) Industrial Use of Medium-Throughput Fragment Screening by Crystal Soaking through Highly Streamlined Sample Preparation at Diamond. Unpublished poster presentation at: Fragments 2015: Fifth RSC-BMCS Fragment-based Drug Discovery meeting. 22 - 24 March 2015, Cambridge, United Kingdom
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