title: Gentle, fast and effective crystal soaking by acoustic dispensing.
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.
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Collins
authors.affiliation: Structural Genomics Consortium (SGC), University of Oxford, Oxford OX3 7DQ, England.
authors.forename: Jia Tsing
authors.initials: JT
authors.lastname: Ng
authors.affiliation: Structural Genomics Consortium (SGC), University of Oxford, Oxford OX3 7DQ, England.
authors.forename: Romain
authors.initials: R
authors.lastname: Talon
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.
authors.forename: Karolina
authors.initials: K
authors.lastname: Nekrosiute
authors.affiliation: Structural Genomics Consortium (SGC), University of Oxford, Oxford OX3 7DQ, England.
authors.forename: Tobias
authors.initials: T
authors.lastname: Krojer
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.
authors.forename: Alice
authors.initials: A
authors.lastname: Douangamath
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.
authors.forename: Jose
authors.initials: J
authors.lastname: Brandao-Neto
authors.affiliation: Structural Genomics Consortium (SGC), University of Oxford, Oxford OX3 7DQ, England.
authors.forename: Nathan
authors.initials: N
authors.lastname: Wright
authors.affiliation: Structural Genomics Consortium (SGC), University of Oxford, Oxford OX3 7DQ, England.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Pearce
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.
authors.forename: Frank
authors.initials: F
authors.lastname: von Delft
mesh_terms: D000162:Acoustics / Q000295:instrumentation*; D003460:Crystallization / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D018360:Crystallography, X-Ray; D004867:Equipment Design; D011506:Proteins / Q000737:chemistry*; D013997:Time Factors
chemical_list: D011506:Proteins
keywords: Diamond Light Source I04-1; Structural Genomics Consortium; XChem; acoustic droplet ejection; crystal soaking; fragment screening