Cargando…

Gentle, fast and effective crystal soaking by acoustic dispensing

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 exp...

Descripción completa

Detalles Bibliográficos
Autores principales: Collins, Patrick M., Ng, Jia Tsing, Talon, Romain, Nekrosiute, Karolina, Krojer, Tobias, Douangamath, Alice, Brandao-Neto, Jose, Wright, Nathan, Pearce, Nicholas M., von Delft, Frank
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349437/
https://www.ncbi.nlm.nih.gov/pubmed/28291760
http://dx.doi.org/10.1107/S205979831700331X
_version_ 1782514467402153984
author Collins, Patrick M.
Ng, Jia Tsing
Talon, Romain
Nekrosiute, Karolina
Krojer, Tobias
Douangamath, Alice
Brandao-Neto, Jose
Wright, Nathan
Pearce, Nicholas M.
von Delft, Frank
author_facet Collins, Patrick M.
Ng, Jia Tsing
Talon, Romain
Nekrosiute, Karolina
Krojer, Tobias
Douangamath, Alice
Brandao-Neto, Jose
Wright, Nathan
Pearce, Nicholas M.
von Delft, Frank
author_sort Collins, Patrick M.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-5349437
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher International Union of Crystallography
record_format MEDLINE/PubMed
spelling pubmed-53494372017-03-24 Gentle, fast and effective crystal soaking by acoustic dispensing Collins, Patrick M. Ng, Jia Tsing Talon, Romain Nekrosiute, Karolina Krojer, Tobias Douangamath, Alice Brandao-Neto, Jose Wright, Nathan Pearce, Nicholas M. von Delft, Frank Acta Crystallogr D Struct Biol Research Papers 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. International Union of Crystallography 2017-03-06 /pmc/articles/PMC5349437/ /pubmed/28291760 http://dx.doi.org/10.1107/S205979831700331X Text en © Collins et al. 2017 http://creativecommons.org/licenses/by/2.0/uk/ This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.http://creativecommons.org/licenses/by/2.0/uk/
spellingShingle Research Papers
Collins, Patrick M.
Ng, Jia Tsing
Talon, Romain
Nekrosiute, Karolina
Krojer, Tobias
Douangamath, Alice
Brandao-Neto, Jose
Wright, Nathan
Pearce, Nicholas M.
von Delft, Frank
Gentle, fast and effective crystal soaking by acoustic dispensing
title Gentle, fast and effective crystal soaking by acoustic dispensing
title_full Gentle, fast and effective crystal soaking by acoustic dispensing
title_fullStr Gentle, fast and effective crystal soaking by acoustic dispensing
title_full_unstemmed Gentle, fast and effective crystal soaking by acoustic dispensing
title_short Gentle, fast and effective crystal soaking by acoustic dispensing
title_sort gentle, fast and effective crystal soaking by acoustic dispensing
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349437/
https://www.ncbi.nlm.nih.gov/pubmed/28291760
http://dx.doi.org/10.1107/S205979831700331X
work_keys_str_mv AT collinspatrickm gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT ngjiatsing gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT talonromain gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT nekrosiutekarolina gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT krojertobias gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT douangamathalice gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT brandaonetojose gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT wrightnathan gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT pearcenicholasm gentlefastandeffectivecrystalsoakingbyacousticdispensing
AT vondelftfrank gentlefastandeffectivecrystalsoakingbyacousticdispensing