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Gentle, fast and effective crystal soaking by acoustic dispensing

DOI: 10.1107/S205979831700331X DOI Help

Authors: Patrick M. Collins (Diamond Light Source) , Jia Tsing Ng (Structural Genomics Consortium (SGC), University of Oxford) , Romain Talon (Structural Genomics Consortium (SGC), University of Oxford) , Karolina Nekrosiute (Diamond Light Source) , Tobias Krojer (Structural Genomics Consortium (SGC), University of Oxford) , Alice Douangamath (Diamond Light Source) , Jose Brandao-Neto (Diamond Light Source) , Nathan Wright (Structural Genomics Consortium (SGC), University of Oxford) , Nicholas M. Pearce (Structural Genomics Consortium, University of Oxford) , Frank Von Delft (Diamond Light Source; Structural Genomics Consortium (SGC), University of Oxford; University of Johannesburg)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Structural Biology , VOL 73 , PAGES 246 - 255

State: Published (Approved)
Published: March 2017

Open Access Open Access

Abstract: The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.

Journal Keywords: fragment screening; crystal soaking; acoustic droplet ejection; Diamond Light Source I04-1; Structural Genomics Consortium; XChem

Subject Areas: Technique Development, Biology and Bio-materials

Diamond Offline Facilities: XChem
Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 16/03/2017 15:28


Discipline Tags:

Technique Development - Life Sciences & Biotech Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX) Fragment Screening